Pyrazolo[3,4-d]pyrimidine derivatives and their use as purinergic receptor antagonists

ABSTRACT

Use of a compound of formula (I): wherein R 1  is selected from alkyl, alkoxy, aryloxy, alkylthio, arylthio, aryl, halogen, CN, NR 6 R 7 , NR 5 COR 6 , NR 5 CONR 6 R 7 , NR 5 CO 2 R 8  and NR 5 SO 2 R 8 ; R 2  is selected from heteroaryl attached via an unsaturated carbon; R 3  is selected from H, alkyl, halogen, OR 5 , SR 5  and NR 6 R 7 ; R 4  is selected from H, acyclic alkyl, CONR 6 R 7 , CONR 5 NR 6 R 7 , COR 6 , CO 2 R 8  and SO 2 R; R 5 , R 6  and R 7  are independently selected from H, alkyl and aryl, or where R 6  and R 7  are in an NR 6 R 7  group, R 6  and R 7  may be linked to form a heterocyclic group, or where R 5 , R 6  and R 7  are in a (CONR 5 NR 6 R 7 ) group, R 5  and R 6  may be linked to form a heterocyclic group; and R 8  is selected from alkyl and aryl, or a pharmaceutically acceptable salt thereof or prodrug thereof, in the treatment or prevention of a disorder in which the blocking of purine receptors, particularly adenosine receptors and more particularly A 2A  receptors, may be beneficial, particularly wherein said disorder is a movement disorder such as Parkinson&#39;s disease or said disorder is depression, cognitive or memory impairment, acute or chronic pain, ADHD or narcolepsy, or for neuroprotection in a subject; compounds of formula (I) for use in therapy; and novel compounds of formula (I) per se.

The present invention relates to pyrazolo[3,4-d]pyrimidine derivatives and their use in therapy. In particular, the present invention relates to the treatment of disorders in which the reduction of purinergic neurotransmission could be beneficial. The invention relates in particular to the blockade of adenosine receptors and particularly adenosine A_(2A) receptors, and to the treatment of movement disorders such as Parkinson's disease.

Movement disorders constitute a serious health problem, especially amongst the elderly sector of the population. These movement disorders are often the result of brain lesions. Disorders involving the basal ganglia which result in movement disorders include Parkinson's disease, Huntington's chorea and Wilson's disease. Furthermore, dyskinesias often arise as sequelae of cerebral ischaemia and other neurological disorders.

There are four classic symptoms of Parkinson's disease: tremor, rigidity, akinesia and postural changes. The disease is also commonly associated with depression, dementia and overall cognitive decline. Parkinson's disease has a prevalence of 1 per 1,000 of the total population. The incidence increases to 1 per 100 for those aged over 60 years. Degeneration of dopaminergic neurones in the substantia nigra and the subsequent reductions in interstitial concentrations of dopamine in the striatum are critical to the development of Parkinson's disease. Some 80% of cells from the substantia nigra need to be destroyed before the clinical symptoms of Parkinson's disease are manifested.

Current strategies for the treatment of Parkinson's disease are based on transmitter replacement therapy (L-dihydroxyphenylacetic acid (L-DOPA)), inhibition of monoamine oxidase (e.g. Deprenyl®), dopamine receptor agonists (e.g. bromocriptine and apomorphine) and anticholinergics (e.g. benztrophine, orphenadrine). Transmitter replacement therapy in particular does not provide consistent clinical benefit, especially after prolonged treatment when “on-off” symptoms develop, and this treatment has also been associated with involuntary movements of athetosis and chorea, nausea and vomiting. Additionally current therapies do not treat the underlying neurodegenerative disorder resulting in a continuing cognitive decline in patients. Despite new drug approvals, there is still a medical need in terms of improved therapies for movement disorders, especially Parkinson's disease. In particular, effective treatments requiring less frequent dosing, effective treatments which are associated with less severe side-effects, and effective treatments which control or reverse the underlying neurodegenerative disorder, are required.

Blockade of A₂ adenosine receptors has recently been implicated in the treatment of movement disorders such as Parkinson's disease (Richardson, P. J. et al., Trends Pharmacol. Sci. 1997, 18, 338–344) and in the treatment of cerebral ischaemia (Gao, Y. and Phillis, J. W., Life Sci. 1994, 55, 61–65). The potential utility of adenosine A_(2A) receptor antagonists in the treatment of movement disorders such as Parkinson's Disease has recently been reviewed (Mally, J. and Stone, T. W., CNS Drugs, 1998, 10, 311–320).

Adenosine is a naturally occurring purine nucleoside which has a wide variety of well-documented regulatory functions and physiological effects. The central nervous system (CNS) effects of this endogenous nucleoside have attracted particular attention in drug discovery, owing to the therapeutic potential of purinergic agents in CNS disorders (Jacobson, K. A. et al., J. Med. Chem. 1992, 35, 407–422). This therapeutic potential has resulted in considerable recent research endeavour within the field of adenosine receptor agonists and antagonists (Bhagwhat, S. S.; Williams, M. Exp. Opin. Ther. Patents 1995, 5,547–558).

Adenosine receptors represent a subclass (P₁) of the group of purine nucleotide and nucleoside receptors known as purinoreceptors. The main pharmacologically distinct adenosine receptor subtypes are known as A₁, A_(2A), A_(2B) (of high and low affinity) and A₃ (Fredholm, B. B., et al., Pharmacol. Rev. 1994, 46, 143–156). The adenosine receptors are present in the CNS (Fredholm, B. B., News Physiol. Sci., 1995, 10, 122–128).

The design of P₁ receptor-mediated agents has been reviewed (Jacobson, K. A., Suzuki, F., Drug Dev. Res., 1997, 39, 289–300; Baraldi, P. G. et al., Curr. Med. Chem. 1995, 2, 707–722), and such compounds are claimed to be useful in the treatment of cerebral ischemia or neurodegenerative disorders, such as Parkinson's disease (Williams, M. and Burnstock, G. Purinergic Approaches Exp. Ther. (1997), 3–26. Editor: Jacobson, Kenneth A.; Jarvis, Michael F. Publisher: Wiley-Liss, New York, N.Y.)

It has been speculated that xanthine derivatives such as caffeine may offer a form of treatment for attention-deficit hyperactivity disorder (ADHD). A number of studies have demonstrated a beneficial effect of caffeine on controlling the symptoms of ADHD (Garfinkel, B. D. et al., Psychiatry, 1981, 26, 395–401). Antagonism of adenosine receptors is thought to account for the majority of the behavioural effects of caffeine in humans and thus blockade of adenosine A_(2A) receptors may account for the observed effects of caffeine in ADHD patients. Therefore a selective A_(2A) receptor antagonist may provide an effective treatment for ADHD but without the unwanted side-effects associated with current therapy.

Adenosine receptors have been recognised to play an important role in regulation of sleep patterns, and indeed adenosine antagonists such as caffeine exert potent stimulant effects and can be used to prolong wakefulness (Porkka-Heiskanen, T. et al., Science, 1997, 276, 1265–1268). Recent evidence suggests that a substantial part of the actions of adenosine in regulating sleep is mediated through the adenosine A_(2A) receptor (Satoh, S., et al., Proc. Natl. Acad. Sci., USA, 1996). Thus, a selective A_(2A) receptor antagonist may be of benefit in counteracting excessive sleepiness in sleep disorders such as hypersomnia or narcolepsy.

It has recently been observed that patients with major depression demonstrate a blunted response to adenosine agonist-induced stimulation in platelets, suggesting that a dysregulation of A_(2A) receptor function may occur during depression (Berk, M. et al, 2001, Eur. Neuropsychopharmacol. 11, 183–186). Experimental evidence in animal models has shown that blockade of A_(2A) receptor function confers antidepressant activity (El Yacoubi, M et al. Br. J. Pharmacol. 2001, 134, 68–77). Thus, A_(2A) receptor antagonists may offer a novel therapy for the treatment of major depression and other affective disorders in patients.

The pharmacology of adenosine A_(2A) receptors has been reviewed (Ongini, E.; Fredholm, B. B. Trends Pharmacol. Sci. 1996, 17(10), 364–372). One potential underlying mechanism in the aforementioned treatment of movement disorders by the blockade of A₂ adenosine receptors is the evidence of a functional link between adenosine A_(2A) receptors to dopamine D₂ receptors in the CNS. Some of the early studies (e.g. Ferre, S. et al., Stimulation of high-affinity adenosine A₂ receptors decreases the affinity of dopamine D₂ receptors in rat striatal membranes. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7238–41) have been summarised in two more recent articles (Fuxe, K. et al., Adenosine Adenine Nucleotides Mol. Biol. Integr. Physiol., [Proc. Int. Symp.], 5th (1995), 499–507. Editors: Belardinelli, Luiz; Pelleg, Amir. Publisher: Kluwer, Boston, Mass.; Ferre, S. et al., Trends Neurosci. 1997, 20, 482–487).

As a result of these investigations into the functional role of adenosine A_(2A) receptors in the CNS, especially in vivo studies linking A₂ receptors with catalepsy (Ferre et al., Neurosci. Lett. 1991, 130, 162–4; Mandhane, S. N. et al., Eur. J. Pharmacol. 1997, 328, 135–141) investigations have been made into agents which selectively bind to adenosine A_(2A) receptors as potentially effective treatments for Parkinson's disease.

While many of the potential drugs for treatment of Parkinson's disease have shown benefit in the treatment of movement disorders, an advantage of adenosine A_(2A) antagonist therapy is that the underlying neurodegenerative disorder may also be treated. The neuroprotective effect of adenosine A_(2A) antagonists has been reviewed (Ongini, E.; Adami, M.; Ferri, C.; Bertorelli, R., Ann. N. Y. Acad. Sci. 1997, 825(Neuroprotective Agents), 30–48). In particular, compelling recent evidence suggests that blockade of A_(2A) receptor function confers neuroprotection against MPTP-induced neurotoxicity in mice (Chen, J-F., J. Neurosci. 2001, 21, RC143). In addition, several recent studies have shown that consumption of dietary caffeine, a known adenosine A_(2A) receptor antagonist, is associated with a reduced risk of Parkinson's disease in man (Ascherio, A. et al, Ann Neurol., 2001, 50, 56–63; Ross G W, et al., JAMA, 2000, 283, 2674–9). Thus, A_(2A) receptor antagonists may offer a novel treatment for conferring neuroprotection in neurodegenerative diseases such as Parkinson's disease.

Xanthine derivatives have been disclosed as adenosine A₂ receptor antagonists as useful for treating various diseases caused by hyperfunctioning of adenosine A₂ receptors, such as Parkinson's disease (see, for example, EP-A-565377).

One prominent xanthine-derived adenosine A_(2A) selective antagonist is CSC [8-(3-chlorostyryl)caffeine] (Jacobson et al., FEBS Lett., 1993, 323, 141–144).

Theophylline (1,3-dimethylxanthine), a bronchodilator drug which is a mixed antagonist at adenosine A₁ and A_(2A) receptors, has been studied clinically. To determine whether a formulation of this adenosine receptor antagonist would be of value in Parkinson's disease an open trial was conducted on 15 Parkinsonian patients, treated for up to 12 weeks with a slow release oral theophylline preparation (150 mg/day), yielding serum theophylline levels of 4.44 mg/L after one week. The patients exhibited significant improvements in mean objective disability scores and 11 reported moderate or marked subjective improvement (Mally, J., Stone, T. W. J. Pharm. Pharmacol. 1994, 46, 515–517).

KF 17837 [(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine] is a selective adenosine A_(2A) receptor antagonist which on oral administration significantly ameliorated the cataleptic responses induced by intracerebroventricular administration of an adenosine A_(2A) receptor agonist, CGS 21680. KF 17837 also reduced the catalepsy induced by haloperidol and reserpine. Moreover, KF 17837 potentiated the anticataleptic effects of a subthreshold dose of L-DOPA plus benserazide, suggesting that KF 17837 is a centrally active adenosine A_(2A) receptor antagonist and that the dopaminergic function of the nigrostriatal pathway is potentiated by adenosine A_(2A) receptor antagonists (Kanda, T. et al., Eur. J. Pharmacol. 1994, 256, 263–268). The structure activity relationship (SAR) of KF 17837 has been published (Shimada, J. et al., Bioorg. Med. Chem. Lett. 1997, 7, 2349–2352). Recent data has also been provided on the A_(2A) receptor antagonist KW-6002 (Kuwana, Y et al., Soc. Neurosci. Abstr. 1997, 23, 119.14; and Kanda, T. et al., Ann. Neurol. 1998, 43(4), 507–513).

New non-xanthine structures sharing these pharmacological properties include SCH 58261 and its derivatives (Baraldi, P. G. et al., Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine Derivatives: Potent and Selective A_(2A) Adenosine Antagonists. J. Med. Chem. 1996, 39, 1164–71). SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) is reported as effective in the treatment of movement disorders (Ongini, E. Drug Dev. Res. 1997, 42(2), 63–70) and has been followed up by a later series of compounds (Baraldi, P. G. et al., J. Med. Chem. 1998, 41(12), 2126–2133).

The foregoing discussion indicates that a potentially effective treatment for movement disorders in humans would comprise agents which act as antagonists at adenosine A_(2A) receptors.

It has now been found that pyrazolo[3,4-d]pyrimidine derivatives, which are structurally unrelated to known adenosine receptor antagonists, exhibit unexpected antagonist binding affinity at adenosine (P₁) receptors, and in particular at the adenosine A_(2A) receptor. Such compounds may therefore be useful for the treatment of disorders in which the blocking of purine receptors, particularly adenosine receptors and more particularly adenosine A_(2A) receptors, may be beneficial. In particular, such compounds may be suitable for the treatment of movement disorders, such as disorders of the basal ganglia which result in dyskinesias. Disorders of particular interest in the present invention include Parkinson's disease, Alzheimer's disease, spasticity, Huntington's chorea and Wilson's disease.

Such compounds may also be particularly suitable for the treatment of depression, cognitive or memory impairment including Alzheimer's disease, acute or chronic pain, ADHD, narcolepsy or for neuroprotection.

According to the present invention there is provided the use of a compound of formula (I):

wherein

-   -   R₁ is selected from alkyl, alkoxy, aryloxy, alkylthio, arylthio,         aryl, halogen, CN, NR₆R₇, NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈ and         NR₅SO₂R₈;     -   R₂ is selected from heteroaryl attached via an unsaturated         carbon of said heteroaryl;     -   R₃ is selected from H, alkyl, halogen, OR₅, SR₅ and NR₆R₇;     -   R₄ is selected from H, acyclic alkyl, CONR₆R₇, CONR₅NR₆R₇, COR₆,         CO₂R₈ and SO₂R₈;     -   R₅, R₆ and R₇ are independently selected from H, alkyl and aryl         or where R₆ and R₇ are in an NR₆R₇ group R₆ and R₇ may be linked         to form a heterocyclic group, or where R₅, R₆ and R₇ are in a         (CONR₅NR₆R₇) group, R₅ and R₆ may be linked to form a         heterocyclic group; and     -   R₈ is selected from alkyl and aryl,         or a pharmaceutically acceptable salt or prodrug thereof, in the         manufacture of a medicament for the treatment or prevention of a         disorder in which the blocking of purine receptors, particularly         adenosine receptors and more particularly A_(2A) receptors, may         be beneficial.

As used herein the term “alkyl”, unless otherwise stated, means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical which may be substituted or unsubstituted. Where cyclic, the alkyl group is preferably C₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅, C₆ or C₇. Where acyclic, the alkyl group is preferably C₁ to C₁₀, more preferably C₁ to C₆, more preferably methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, isobutyl or tertiary-butyl) or pentyl (including n-pentyl and isopentyl), more preferably methyl. It will be appreciated therefore that the term “alkyl” as used herein, unless otherwise stated, includes alkyl (branched or unbranched), alkenyl (branched or unbranched), alkynyl (branched or unbranched), cycloalkyl, cycloalkenyl and cycloalkynyl.

As used herein, the term “lower alkyl” means methyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl, isobutyl or tertiary-butyl).

As used herein, the term “aryl” means an aromatic group, such as phenyl or naphthyl (preferably phenyl), or a heteroaromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, isoxazolyl, pyrazolyl, triazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl and indazolyl.

As used herein, the term “heteroaryl” means an aromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, isoxazolyl, pyrazolyl, triazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl and indazolyl.

As used herein, the term “non-aromatic heterocyclyl” means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a cyclic amino group (including aziridinyl, azetidinyl, pyrrolidinyl, piperidyl, piperazinyl, morpholinyl) or a cyclic ether (including tetrahydrofuranyl).

As used herein, the term “alkoxy” means alkyl-O—. As used herein, the term “aryloxy” means aryl-O—.

As used herein, the term “halogen” means a fluorine, chlorine, bromine or iodine radical.

As used herein, the term “ortho,ortho-disubstituted aryl groups” refers to heteroaryl groups which are substituted in both ortho positions of the heteroaryl group relative to the point of attachment of the heteroaryl group to the pyrimidine ring.

As used herein, the term “prodrug” means any pharmaceutically acceptable prodrug of a compound of the present invention.

Where any of R₁ to R₁₄ is selected from alkyl, alkoxy and thioalkyl, in accordance with formula (I) as defined above, then that alkyl group, or the alkyl group of the alkoxy or thioalkyl group, may be substituted or unsubstituted. Where any of R₁ to R₁₄ are selected from aryl, aryloxy and thioaryl, in accordance with formula (I) as defined above, then said aryl group, or the aryl group of the aryloxy or thioaryl group, may be substituted or unsubstituted. Where R₆ and R₇, or R₅ and R₆, are linked to form a heterocyclic group, the heterocyclic group may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. Substituents may include:

-   carbon-containing groups such as     -   alkyl,     -   aryl, (e.g. substituted and unsubstituted phenyl (including         (alkyl)phenyl, (alkoxy)phenyl, (alkyl- and         aryl-sulfonylamino)phenyl and halophenyl),     -   arylalkyl; (e.g. substituted and unsubstituted benzyl); -   halogen atoms and halogen containing groups such as     -   haloalkyl (e.g. trifluoromethyl),     -   haloaryl (e.g. chlorophenyl); -   oxygen containing groups such as     -   alcohols (e.g. hydroxy, hydroxyalkyl, hydroxyaryl,         (aryl)(hydroxy)alkyl),     -   ethers (e.g. alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl,         alkoxyaryl, aryloxyaryl),     -   aldehydes (e.g. carboxaldehyde),     -   ketones (e.g. alkylcarbonyl, arylcarbonyl, alkylcarbonylalkyl,         alkylcarbonylaryl, arylcarbonylalkyl, arylcarbonylaryl,         arylalkylcarbonyl, arylalkylcarbonylalkyl,         arylalkylcarbonylaryl)     -   acids (e.g. carboxy, carboxyalkyl, carboxyaryl),     -   acid derivatives such as esters (e.g. alkoxycarbonyl,         aryloxycarbonyl, alkoxycarbonylalkyl, aryloxycarbonylalkyl,         alkoxycarbonylaryl, aryloxycarbonylaryl, alkylcarbonyloxy,         alkylcarbonyloxyalkyl), amides     -   (e.g. aminocarbonyl, mono- or di-alkylaminocarbonyl,         cyclicaminocarbonyl, aminocarbonylalkyl, mono- or         di-alkylaminocarbonylalkyl, arylaminocarbonyl or         arylalkylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino or         arylalkylcarbonylamino), carbamates     -   (eg. alkoxycarbonylamino, aryloxycarbonylamino,         arylalkyloxycarbonylamino, aminocarbonyloxy, mono- or         di-alkylaminocarbonyloxy, arylaminocarbonyloxy or         arylalkylaminocarbonyloxy) and ureas (eg. mono- or         di-alkylaminocarbonylamino, arylaminocarbonylamino or         arylalkylaminocarbonylamino); -   nitrogen containing groups such as     -   amines (e.g. amino, mono- or dialkylamino, cyclicamino,         arylamino, aminoalkyl, mono- or dialkylaminoalkyl),     -   azides,     -   nitriles (e.g. cyano, cyanoalkyl),     -   nitro,     -   sulfonamides (e.g. aminosulfonyl, mono- or         di-alkylaminosulfonyl, mono- or di-arylaminosulfonyl, alkyl- or         aryl-sulfonylamino, alkyl- or aryl-sulfonyl(alkyl)amino, alkyl-         or aryl-sulfonyl(aryl)amino); -   sulfur containing groups such as     -   thiols, thioethers, sulfoxides, and sulfones (e.g. alkylthio,         alkylsulfinyl, alkylsulfonyl, alkylthioalkyl,         alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl,         arylsulfonyl, arylthioalkyl, arylsulfinylalkyl,         arylsulfonylalkyl) -   and heterocyclic groups containing one or more, preferably one,     heteroatom, (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl,     thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl,     aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl,     imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl,     pyridyl, pyrazinyl, pyridazinyl, piperidyl, hexahydroazepinyl,     piperazinyl, morpholinyl, thianaphthyl, benzofuranyl,     isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl,     indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl,     quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl, quinazolinyl,     pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl,     isochromanyl, phthalazinyl and carbolinyl).

Where any of R₁ to R₁₄ is selected from aryl or from an aryl-containing group such as aryloxy or arylthio, preferred substituent group(s) are selected from halogen, alkyl (substituted or unsubstituted; and where substituted particularly from alkoxyalkyl, hydroxyalkyl, aminoalkyl and haloalkyl), hydroxy, alkoxy, CN, NO₂, amines (including amino, mono- and di-alkylamino), alkoxycarbonyl, aminocarbonyl, carboxamido, sulfonamido, alkoxycarbonylamino and aryl, and particularly from unsubstituted alkyl, substituted alkyl (including alkoxyalkyl and aminoalkyl), halogen and amines.

In one embodiment, where any of R₁ to R₁₄ is directly substituted by an alkyl substituent group, or by an alkyl-containing substituent group (such as alkoxy or alkylcarbonylamino for example), then the alkyl moiety of the substituent group directly attached to any of R₁ to R₁₄ may be further substituted by the substituent groups hereinbefore described and particularly by halogen, hydroxy, alkoxy, CN, amines (including amino, mono- and di-alkyl amino) and aryl.

In a further embodiment, where any of R₁ to R₁₄ is directly substituted by an aryl substitutent group, or by an arylcontaining substituent group (such as aryloxy or arylaminocarbonylamino for example), then the aryl moiety of the substituent group directly attached to any of R₁ to R₁₄ may be further substituted by the substituent groups hereinbefore described and particularly by halogen, alkyl (substituted or unsubstituted; and where substituted particularly from alkoxyalkyl, hydroxyalkyl, aminoalkyl and haloalkyl), hydroxy, alkoxy, CN, NO₂, amines (including amino, mono- and di-alkylamino), alkoxycarbonyl, aminocarbonyl, carboxamido, sulfonamido, alkoxycarbonylamino and aryl. In a further embodiment, said aryl moiety is substituted by halogen, alkyl (including CF₃), hydroxy, alkoxy, CN, amines (including amino, mono- and di-alkyl amino) and NO₂. In a further embodiment, said aryl moiety is substituted by unsubstituted alkyl, substituted alkyl (particularly alkoxyalky and aminoalkyl), halogen and amines.

The terms “directly substituted” and “directly attached”, as used herein, mean that the substituent group is bound directly to any of R₁ to R₁₄ without any intervening divalent atoms or groups.

In the compounds of formula (I), R₁ is selected from alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, and cyclic and acyclic alkyl), alkoxy, aryloxy, alkylthio, arylthio, aryl (including heteroaryl), halogen, CN, NR₆R₇ (including NH₂, mono-alkylamino and di-alkylamino), NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈ and NR₅SO₂R₈.

Where R₁ is selected from alkyl, alkoxy and alkylthio, then said alkyl group or the alkyl group of the alkoxy or alkylthio is preferably selected from C₁₋₆ alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, and cyclic and acyclic alkyl), preferably saturated C₁₋₆ alkyl, and more preferably lower alkyl. In one embodiment, R₁ is selected from substituted alkyl, particularly haloalkyl (including CF₃) and arylalkyl (including heteroarylalkyl), and particularly haloalkyl (including CF₃).

Where R₁ is selected from NR₅CONR₆R₇, NR₅COR₆, NR₅CO₂R₈ and NR₅SO₂R₈, preferably R₅ is H or alkyl, and preferably H.

It is preferred that R₁ is selected from alkyl, alkoxy, thioalkyl, NR₆R₇ (including NH₂), NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈ and NR₅SO₂R₈, and preferably from NR₆R₇ (including NH₂), NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈ and NR₅SO₂R₈, more preferably from NR₆R₇ (including NH₂), and more preferably from NH₂.

In the compounds of formula (I), R₂ is substituted or unsubstituted heteroaryl attached via an unsaturated carbon atom. Preferably, the heteroaryl group is a 5- or 6-membered monocyclic group.

Preferably, R₂ is a heteroaryl group which is attached to the pyrimidine ring of formula (I) such that at least one heteroatom is adjacent to the unsaturated carbon atom attached to the pyrimidine ring. Preferably, R₂ is an N, O or S-containing heteroaryl group. R₂ may contain one or more heteroatom(s) selected from N, O and S.

It is preferred that R₂ is not an ortho,ortho-disubstituted heteroaryl group, and preferably R₂ is not substituted at either ortho position. As used herein, reference to ortho-substitution of the R₂ group means the ortho positions of the R₂ group relative to the point of attachment of R₂ to the pyrimidine moiety of formula (I).

In a preferred embodiment, R₂ is selected from furyl (including 2-furyl), thienyl (including 2-thienyl), pyridyl (including 2-pyridyl), thiazolyl (including 2- and 5-thiazolyl), pyrazolyl (including 3-pyrazolyl), triazolyl (including 4-triazolyl), pyrrolyl (including 2-pyrrolyl) and oxazolyl (including 5-oxazolyl). In a further embodiment, R₂ is selected from 2-furyl, 2-thienyl, 2-thiazolyl, 2-pyridyl, 3-pyrazolyl, 2-pyrrolyl, 4-triazolyl and 5-oxazolyl. In a further preferred embodiment, R₂ is selected from furyl, thienyl, pyridyl, thiazolyl and pyrazolyl, and particularly from 2-furyl, 2-thienyl, 2-thiazolyl, 2-pyridyl and 3-pyrazolyl. In a further embodiment, R₂ is selected from furyl, thienyl and pyridyl, preferably 2-furyl, 2-thienyl and 2-pyridyl. In a particularly preferred embodiment, R₂ is selected from furyl, and preferably from 2-furyl, substituted or unsubstituted.

In the compounds of formula (I), R₃ is selected from H, alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, and cyclic and acyclic alkyl), halogen, OR₅, SR₅ and NR₆R₇.

Where R₃ is selected from alkyl, alkoxy and alkylthio, then said alkyl group or the alkyl group of the alkoxy or alkylthio is preferably selected from C₁₋₆ alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, and cyclic and acyclic alkyl), preferably saturated C₁₋₆ alkyl, and more preferably lower alkyl.

Preferably, R₃ is hydrogen.

In the compounds of formula (I), R₄ is selected from H, acyclic alkyl (including branched and unbranched acyclic alkyl, and substituted and unsubstituted acyclic alkyl), CONR₆R₇, CONR₅NR₆R₇, COR₆, CO₂R₈ and SO₂R₈.

Where R₄ is selected from acyclic alkyl, R₄ is preferably C₁₋₆ acyclic alkyl (including alkenyl and alkynyl). In one embodiment, R₄ is selected from C₁₋₆ saturated acyclic alkyl, preferably lower alkyl.

In one embodiment, R₄ is selected from substituted acyclic alkyl (including saturated acyclic alkyl and alkenyl). Preferred substituents are susbtituted and unsubstituted aryl (including heteroaryl), cycloalkyl, non-aromatic heterocyclyl, CO₂R₅, CONR₆R₇, CONR₅NR₆R₇ and C(═NR₅)NR₆R₇, preferably aryl (including heteroaryl) and CONR₆R₇, more preferably aryl (including heteroaryl).

Where R₄ is selected from acyclic alkyl substituted by aryl (including heteroaryl), the aryl (including heteroaryl) group may unsubstituted, or substituted as defined in more detail below in respect of of the group referred to as R₁₁. Preferably, the aryl-substituted acyclic alkyl is an aryl-substituted methyl group. Preferred aryl groups are set out in detail below in respect of the group referred to as Ar.

In one embodiment, R₄ is selected from (CR₉R₁₀)_(n)R₁₁ wherein n is 1 to 6 (preferably n is 1, 2 or 3, more preferably n is 1), R₉ and R₁₀ are independently selected from H, alkyl and aryl, and R₁₁ is selected from aryl (including heteroaryl), cycloalkyl, non-aromatic heterocyclic, CO₂R₅, CONR₆R₇, CONR₅NR₆R₇ and C(═NR₅)NR₆R₇, wherein R₁₁ is preferably aryl (including heteroaryl) or CONR₆R₇. More preferably R₁₁ is aryl (including heteroaryl). Preferably R₉ and R₁₀ are independently selected from H and alkyl, more preferably H.

Where R₁₁ is selected from CONR₆R₇, R₆ and R₇ are selected from H, alkyl (including substituted alkyl such as arylalkyl (including heteroarylalkyl)) and aryl (including heteroaryl) or R₆ and R₇ may be linked to form a heterocyclic ring. In one embodiment, R₆ and R₇ are selected from H, unsubstituted alkyl, arylalkyl (including heteroarylalkyl) and aryl (including heteroaryl). Said aryl groups may be substituted or unsubstituted. In a preferred embodiment one of R₆ and R₇ is hydrogen.

Where R₁₁ is aryl (including heteroaryl), the aryl (including heteroaryl) group may be unsubstituted, or may be substituted. In one embodiment, R₁₁ is selected from the group consisting of substituted aryl (including heteroaryl) groups, preferably mono-, di- or tri-substituted aryl (including heteroaryl) groups represented by the formula Ar(R₁₂)_(a)(R₁₃)_(b)(R₁₄)_(c) wherein Ar is an aryl (including heteroaryl) group; wherein R₁₂, R₁₃ and R₁₄ are substituent group(s), the same or different; and wherein a, b and c are 0 or 1 such that a+b+c≧1.

In one embodiment, the group Ar is selected from phenyl. In an alternative embodiment, the group Ar is selected from heteroaryl groups such as those described hereinabove, preferably from mono or bicyclic heteroaryl groups, more preferably from pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl, preferably 2-pyridyl), indolyl (including 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl and 7-indolyl), furyl (including 2-furyl and 3-furyl, preferably 2-furyl), thienyl (including 2-thienyl and 3-thienyl, preferably 2-thienyl), isoindolyl, indolinyl, isoxazolyl, oxazolyl, thiazolyl, pyrazinyl, pyrimidinyl, quinolinyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, indazolyl, benzodioxolyl and dihydrobenzofuranyl, more preferably from pyridyl (preferably 2-pyridyl), indolyl, furyl (preferably 2-furyl) and thienyl (preferably 2-thienyl), and most preferably from pyridyl (preferably 2-pyridyl), furyl (preferably 2-furyl) and thienyl (preferably 2-thienyl).

In one embodiment, the group Ar is selected from phenyl, pyridyl (preferably 2-pyridyl), furyl (preferably 2-furyl), thienyl (preferably 2-thienyl) and indolyl, and particularly from phenyl, pyridyl (preferably 2-pyridyl), furyl (preferably 2-furyl) and thienyl (preferably 2-thienyl).

The substituent groups R₁₂, R₁₃ and R₁₄ may be selected from any of the substituent groups described herein above.

In a preferred embodiment, R₁₂, R₁₃ and R₁₄ are selected from NR₆R₇ (including NH₂, and NHR₆), alkyl (substituted or unsubstituted; preferably C₁₋₆ acyclic alkyl), alkoxy (including fluoroalkoxy), halogen (including F, Cl, Br and I), NO₂, CN, hydroxy, NHOH, CHO, CONR₆R₇, CO₂R₅, NR₅COR₆ (preferably NHCOR₆), NR₅CO₂R₈ (preferably NHCO₂R₈), NR₅SO₂R₈ (preferably NHSO₂R₈), OCO₂R₈ and aryl (including heteroaryl).

In a more preferred embodiment, R₁₂, R₁₃ and R₁₄ are selected from NR₆R₇ (including NH₂ and NHR₆), alkyl (substituted or unsubstituted; and preferably C₁₋₆ acyclic saturated alkyl) and halogen (preferably F or Cl, particularly F).

In a particularly preferred embodiment, R₁₂, R₁₃ and R₁₄ are selected from NR₆R₇ (including NH₂ and NHR₆, preferably NH₂) and alkyl (substituted or unsubstituted; preferably C₁₋₆ acyclic saturated alkyl;

Where R₁₂, R₁₃ and R₁₄ are selected from substituted alkyl, said alkyl is preferably selected from alkoxyalkyl, hydroxyalkyl, aminoalkyl (including NH₂-alkyl, mono-alkylaminoalkyl and di-alkylaminoalkyl), haloalkyl particularly fluoroalkyl (including CF₃)), cyanoalkyl, alkylthioalkyl, alkylcarboxyaminoalkyl, alkoxycarbonylaminoalkyl and alkylsulfonylamino, more preferably from alkoxyalkyl, hydroxyalkyl, aminoalkyl and haloalkyl (particularly fluoroalkyl (including CF₃)) and most preferably from alkoxyalkyl and aminoalkyl.

In one embodiment, the substituent groups R₁₂, R₁₃ and R₁₄ are selected from halogen, alkyl (including CF₃), hydroxy, alkoxy, alkylthio, CN, amines (including amino, mono- and di-alkyl amino) and NO₂.

Where the Ar group is phenyl, the phenyl ring may be mono-, di- or tri-substituted, preferably wherein the substituent group is selected from NR₆R₇, alkyl, alkoxy, halogen, NO₂, CN, hydroxy, CONR₆R₇, CO₂R₅, NR₅COR₆, NR₅CO₂R₈, NR₅SO₂R₈ and OCO₂R₈, as described above, and more preferably from NR₆R₇ (including NH₂ and NHR₆, and preferably NH₂), alkyl (substituted or unsubstituted; preferably C₁₋₆ acyclic saturated alkyl; and, where substituted, preferably from alkoxyalkyl, hydroxyalkyl, aminoalkyl and haloalkyl particularly fluoroalkyl (including CF₃)), and more preferably from alkoxyalkyl and aminoalkyl) and halogen (preferably F or Cl, particularly F). Where (a+b+c) is 2 or 3, it is preferred that at least one of the substituent groups is NR₆R₇, particularly NH₂.

Where the Ar group is pyridyl, the pyridyl group (which is preferably a 2-pyridyl group) is preferably mono-substituted, preferably 6-substituted. The preferred substituent group(s) are selected from alkyl (including substituted and unsubstituted, saturated and unsaturated (such as alkenyl, including vinyl); and preferably C₁₋₆ acyclic alkyl), alkoxy, halogen, aryl, NO₂, NHOH and CHO, as described above, and more preferably from alkyl (substituted or unsubstituted; preferably C₁₋₆ acyclic saturated alkyl; and, where substituted, preferably from alkoxyalkyl, hydroxyalkyl, aminoalkyl and haloalkyl (particularly fluoroalkyl (including CF₃)), and more preferably from alkoxyalkyl and aminoalkyl).

Where R₄ is selected from CONR₆R₇, R₆ and R₇ are selected from H, alkyl (including substituted alkyl such as arylalkyl (including heteroarylalkyl)) and aryl (including heteroaryl) or R₆ and R₇ may be linked to form a heterocyclic ring. In one embodiment, R₆ and R₇ are selected from H. unsubstituted alkyl, arylalkyl (including heteroarylalkyl) and aryl (including heteroaryl). Said aryl groups may be substituted or unsubstituted. In a preferred embodiment one of R₆ and R₇ is hydrogen. In a further preferred embodiment, R₆ is H and R₇ is selected from arylalkyl (including heteroarylalkyl),preferably arylmethyl (including heteroarylmethyl).

In a preferred embodiment, R₄ is selected from H and substituted acylic alkyl, preferably wherein said acyclic alkyl is substituted by aryl (including heteroaryl) or CONR₆R₇, and preferably by aryl (including heteroaryl), as described herein.

In the compounds of formula (I), R₅, R₆ and R₇ are independently selected from H, alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, cyclic and acyclic alkyl) and aryl (including heteroaryl) or where R₆ and R₇ are in any NR₆R₇ group R₆ and R₇ may be linked to form a heterocyclic group, or where R₅, R₆ and R₇ are in a (CONR₅NR₆R₇) group, R₅ and R₆ may be linked to form a heterocyclic group.

In the compounds of formula (I), R₈ is selected from alkyl (including branched and unbranched alkyl, substituted and unsubstituted alkyl, cyclic and acyclic alkyl) and aryl (including heteroaryl).

Where R₅ to R₈ are independently selected from alkyl, preferably R₅ to R₈ are selected from C₁₋₆ alkyl, preferably C₁₋₆ saturated alkyl and more preferably from lower alkyl.

Where R₆ and R₇, or R₅ and R₆, are linked to form a heterocyclic ring said heterocyclic ring may be saturated, partially unsaturated or aromatic, and is preferably saturated. Said heterocyclic ring is preferably a 5, 6 or 7-membered ring, preferably a 5 or 6-membered ring, and may contain one or more further heteroatom(s) preferably selected from N, O and S.

In a preferred embodiment, R₁ is NH₂, R₂ is 2-furyl, R₃ is H and R₄ is arylmethyl (including heteroarylmethyl).

In a particularly preferred embodiment of the invention, the compounds of formula (I) are selected from:

-   4-(2-furyl)-1-(3-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; -   1-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; -   4-(2-furyl)-1-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; -   1-(2,6-difluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; -   4-(2-furyl)-1-(3-phenylpropyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine;     and -   1-(2-fluorobenzyl)-4-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine.

Where chiral the compounds of formula (I) may be in the form of a racemic mixture of pairs of enantiomers or in enantiomerically pure form.

According to a further aspect of the present invention there is provided a method of treating or preventing a disorder in which the blocking of purine receptors, particularly adenosine receptors and more particularly adenosine A_(2A) receptors, may be beneficial, the method comprising administration to a subject in need of such treatment an effective dose of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.

The disorder may be caused by the hyperfunctioning of the purine receptors.

The present invention may be employed in respect of a human or animal subject, more preferably a mammal, more preferably a human subject.

The disorders of particular interest are those in which the blocking of purine receptors, partiucularly adenosine receptors and more particularly adenosine A_(2A) receptors, may be beneficial. These may include movement disorders such as Parkinson's disease, drug-induced Parkinsonism, post-encephalitic Parkinsonism, Parkinsonism induced by poisoning (for example MPTP, manganese, carbon monoxide) and post-traumatic Parkinson's disease (punch-drunk syndrome).

Other movement disorders in which the blocking of purine receptors, may be of benefit include progressive supernuclear palsy, Huntingtons disease, multiple system atrophy, corticobasal degeneration, Wilsons disease, Hallerrorden-Spatz disease, progressive pallidal atrophy, Dopa-responsive dystonia-Parkinsonism, spasticity or other disorders of the basal ganglia which result in abnormal movement or posture. The present invention may also be effective in treating Parkinson's with on-off phenomena; Parkinson's with freezing (end of dose deterioration); and Parkinson's with prominent dyskinesias.

The compounds of formula (I) may be used or administered in combination with one or more additional drugs useful in the treatment of movement disorders, such as L-DOPA or a dopamine agonist, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.

Other disorders in which the blocking of purine receptors, particularly adenosine receptors and more particularly adenosine A_(2A) receptors may be beneficial include acute and chronic pain; for example neuropathic pain, cancer pain, trigeminal neuralgia, migraine and other conditions associated with cephalic pain, primary and secondary hyperalgesia, inflammatory pain, nociceptive pain, tabes dorsalis, phantom limb pain, spinal cord injury pain, central pain, post-herpetic pain and HIV pain; affective disorders including mood disorders such as bipolar disorder, seasonal affective disorder, depression, manic depression, atypical depression and monodepressive disease; central and peripheral nervous system degenerative disorders including corticobasal degeneration, demyelinating disease (multiple sclerosis, disseminated sclerosis), Freidrich's ataxia, motoneurone disease (amyotrophic lateral sclerosis, progressive bulbar atrophy), multiple system atrophy, myelopathy, radiculopathy, peripheral neuropathy (diabetic neuropathy, tabes dorsalis, drug-induced neuropathy, vitamin deficiency), systemic lupus erythamatosis, granulomatous disease, olivo-ponto-cerebellar atrophy, progressive pallidal atrophy, progressive supranuclear palsy, spasticity; schizophrenia and related pyshoses; cognitive disorders including dementia, Alzheimers Disease, Frontotemporal dementia, multi-infarct dementia, AIDS dementia, dementia associated with Huntingtons Disease, Lewy body dementia, senile dementia, age-related memory impairment, cognitive impairment associated with dementia, Korsakoff syndrome, dementia pugilans; attention disorders such as attention-deficit hyperactivity disorder (ADHD), attention deficit disorder, minimal brain dysfunction, brain-injured child syndrome, hyperkinetic reaction childhood, and hyperactive child syndrome; central nervous system injury including traumatic brain injury, neurosurgery (surgical trauma), neuroprotection for head injury, raised intracranial pressure, cerebral oedema, hydrocephalus, spinal cord injury; cerebral ischaemia including transient ischaemic attack, stroke (thrombotic stroke, ischaemic stroke, embolic stroke, haemorrhagic stroke, lacunar stroke) subarachnoid haemorrhage, cerebral vasospasm, neuroprotection for stroke, peri-natal asphyxia, drowning, cardiac arrest, subdural haematoma; myocardial ischaemia; muscle ischaemia; sleep disorders such as hypersomnia and narcolepsy; eye disorders such as retinal ischaemia-reperfusion injury and diabetic neuropathy; cardiovascular disorders such as claudication and hypotension; and diabetes and its complications.

According to a further aspect of the present invention there is provided use of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for the treatment or prevention of movement disorders in a subject.

According to a further aspect of the invention there is provided a method of treating or preventing movement disorders comprising administration to a subject in need of such treatment an effective dose of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.

According to a further aspect of the invention there is provided use of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof in the manufacture of a medicament for neuroprotection in a subject.

According to a further aspect of the invention there is provided a method of neuroprotection comprising administration to a subject in need of such treatment an effective dose of a compound of formula (I) or a pharmaceutically acceptable salt or prodrug thereof.

The medicament for or method of neuroprotection may be of use in the treatment of subjects who are suffering from or at risk from a neurodegenerative disorder, such as a movement disorder.

According to a further aspect of the invention, there is provided for use in therapy a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof.

According to a further aspect of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof, per se, other than compounds wherein R₂ is selected from pyrazolopyrimidines. In a preferred embodiment, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof, per se, wherein R₂ is selected from 5- or 6-membered monocyclic heteroaryl groups.

According to a further aspect of the invention, there is provided a method of preparing the novel compounds of the present invention. Compounds of formula (I) may be prepared according to conventional synthetic methods, such as set out in Reaction Scheme 1.

Compounds of formula (1) where R₄ is alkyl or substituted alkyl (including arylalkyl, heteroalkylaryl and (CR₉R₁₀)_(n)R₁₁ wherein R₁₁ is CO₂R₅ or CN) may be prepared from compounds of formula (2) by standard methods such as reaction with an appropriate alkyl halide or substituted alkyl halide in the presence of a suitable base such as NaH.

Compounds of formula (1) where R₄ is (CR₉R₁₀)_(n)R₁₁ wherein R₁₁ is CONR₆R₇ or CONR₅NR₆R₇ may be prepared from compounds of formula (1) where R₄ is (CR₉R₁₀)_(n)R₁₁ wherein R₁₁ is CO₂R₅ by standard methods such as direct reaction with an appropriate amine or hydrazine derivative or by initial hydrolysis of the ester group CO₂R₅ to a carboxylic acid followed by reaction with an appropriate amine or hydrazine derivative in the presence of a standard coupling reagent such as DCC.

Compounds of formula (1) where R₄ is (CR₉R₁₀)_(n)R₁₁ wherein R₁₁ is C(═NR₅)NR₆R₇ may be prepared from compounds of formula (1) where R₄ is (CR₉R₁₀)_(n)R₁₁ wherein R₁₁ is CN by standard methods such as treatment with an appropriate amine in the presence of trimethylaluminium.

Compounds of formula (1) where R₄ is CONR₆R₇, wherein one of R₆ or R₇ is H, are prepared from compounds of formula (2) by standard methods such as reaction with an appropriate isocyanate (R₆NCO or R₇NCO). Compounds of formula (1) where R₄ is CONR₆R₇, wherein R₆ and R₇ are alkyl or aryl or are linked to form a heterocyclic group, are prepared from compounds of formula (2) by standard methods such as reaction with an appropriate carbamoyl chloride (R₆R₇NCOCl) in the presence of a suitable base such as Et₃N.

Compounds of formula (1) wherein R₄ is CONR₅NR₆R₇ are prepared from compounds of formula (2) by standard methods such as reaction with an appropriate carbamoyl chloride R₆R₇NR₅NCOCl in the presence of a suitable base such as Et₃N.

Compounds of formula (1) where R₄ is COR₆ or SO₂R₈ are prepared from compounds of formula (2) by standard methods such as reaction with an appropriate acid chloride (ClCOR₆) or sulphonyl chloride (ClSO₂R₈) in the presence of a suitable base such as Et₃N.

Compounds of formula (1) where R₄ is CO₂R₈ are prepared from compounds of formula (2) by standard methods such as reaction with an appropriate chloroformate (ClCO₂R₈) or dicarbonate (O(CO₂R₈)₂) in the presence of a suitable base, if required, such as Et₃N and in the presence of a suitable catalyst, if required, such as DMAP.

Alternatively compounds of formula (1) are prepared from compounds of formula (3) by standard methods such as aryl coupling reactions. Suitable aryl coupling reactions would include reaction of a compound of formula (3) with a heteroaryl trialkylstannane, a heteroarylboronic acid or boronic ester, or a heteroaryl zinc halide reagent in the presence of a suitable catalyst such as a palladium catalyst. Suitable heteroaryl trialkylstannane, heteroarylboronic acid or boronic ester, or heteroaryl zinc halide reagents are either commercially available, known in the literature or may be synthesised by standard methods analogous to those described in the literature. In certain cases it may be advantageous to use a protecting group to protect vulnerable functionality which may be present in the heteroaryl coupling partner. This would be particularly appropriate where the heteroaryl group R₂ is imidazolyl, pyrazolyl or triazolyl where protection of an unsubstituted ring nitrogen may be necessary to facilitate the coupling reaction. Suitable protecting groups in such cases would include trimethylsilylethoxymethyl which can be introduced and removed at a convenient point in the synthesis by standard methods.

Compounds of formula (2) are prepared from compounds of formula (4) by standard methods such as aryl coupling reactions as described above. Compounds of formula (4) where R₁ is alkyl, aryl, alkylthio, halogen and NR₆R₇ are either known in the literature or are prepared by methods analogous to those described in the literature.

Compounds of formula (3) are prepared from compounds of formula (4) by standard methods such as those described above. Compounds of formula (3) where R₁ is alkyl, aryl, alkylthio, halogen or NR₆R₇ are either known in the literature or may be prepared by methods analogous to those described in the literature.

Compounds of formula (1) may alternatively be prepared by standard synthetic methods such as those illustrated in Reaction Scheme 2.

Compounds of formula (1) where R₁ is NR₆R₇, alkoxy, aryloxy, alkylthio, arylthio or CN are prepared from compounds of formula (5) by standard methods such as treatment with an appropriate nucleophile such as an amine (HNR₆R₇), alcohol, thiol or cyanide in the presence of a suitable base if required.

Compounds of formula (5) are prepared from compounds of formula (6) by standard methods such as aryl coupling reactions as described above.

Compounds of formula (6) are either known in the literature or are prepared by methods analogous to those described in the literature. For example compounds of formula (6) are prepared from compounds of formula (7) by standard methods such as treatment with a chlorinating reagent such as POCl₃.

Compounds of formula (7) are either known in the literature or are prepared by methods analogous to those described in the literature. For example compounds of formula (7) are prepared from compounds of formula (8) by standard methods such as heating in the presence of urea. Compounds of formula (8) are either known in the literature or are prepared by methods analogous to those described in the literature.

Compounds of formula (1) where R₁ is NR₅COR₆, NR₅CO₂R₈ or NR₅SO₂R₈ wherein R₅ is H, alkyl or aryl, are prepared from compounds of formula (1) where R₁ is NR₆R₇, wherein one of R₆ or R₇ is H and the other is alkyl or aryl, by standard methods such as reaction with an appropriate acid chloride (ClCOR₆), chloroformate (ClCO₂R₈) or sulphonyl chloride (ClSO₂R₈).

Compounds of formula (1) where R₁ is NR₅CONR₆R₇ wherein R₅ is H, alkyl or aryl, are prepared from compounds of formula (1) where R₁ is NR₆R₇, wherein one of R₆ or R₇ is H and the other is alkyl or aryl, by standard methods such as reaction with an appropriate isocyanate (R₆NCO or R₇NCO) or an appropriate carbamoyl chloride (R₆R₇NCOCl).

Compounds of formula (1) where R₃ is H, alkyl, halogen, OR₅, SR₅ or NR₆R₇ may be prepared by the methods described above. Suitable intermediates such as compounds of formula (3) and (4) where R₃ is as described above, are either known in the literature or are prepared by methods which are analogous to those described in the literature.

In the compounds of the present invention, where any of the groups R₁ to R₈ is an alkyl group or aryl group or where any of the groups R₁ to R₈ contains an alkyl or aryl substituent, the alkyl or aryl group may also be substituted. It will be appreciated by those skilled in the art that certain substituents on the alkyl or aryl groups mentioned above may be introduced directly as an integral part of the substituent R₁ to R₈ by using the synthetic methods described above. In other cases it may be advantageous to introduce certain substituents on the alkyl or aryl groups mentioned above by chemical transformation of other substituent groups. For example where the alkyl or aryl group mentioned above contains an amino substituent this may be converted by standard methods to an alkylamino or dialkylamino group by standard methods such as alkylation or reductive alkylation, or to an amide, carbamate, urea or sulphonamide by standard methods such as those described above. Additionally, for example, where the alkyl or aryl group mentioned above contains a carboxylic ester substituent this may be converted to an amide or hydrazide derivative by standard methods such as reaction with an amine or hydrazine directly or in the presence of a catalyst such as Me₃Al if required. It will be appreciated by those skilled in the art that substituents such as an amino group or a carboxylic ester group may also be transformed by standard methods to a wide range of additional substituent groups.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula (I) in combination with a pharmaceutically acceptable carrier or excipient and a method of making such a composition comprising combining a compound of formula (I) with a pharmaceutically acceptable carrier or excipient.

The pharmaceutical compositions employed in the present invention comprise a compound of formula (I), or pharmaceutically acceptable salts or prodrugs thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients known to those skilled in the art. The term, “pharmaceutically acceptable salts”, refers to salts prepared from pharmaceutically acceptable non-toxic acids including inorganic acids and organic acids.

Where the compounds of formula (I) are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Particularly preferred are hydrochloric, hydrobromic, phosphoric, and sulfuric acids, and most particularly preferred is the hydrochloride salt.

Any suitable route of administration may be employed for providing the patient with an effective dosage of a compound of formula (I). For example, oral, rectal, parenteral (intravenous, intramuscular), transdermal, subcutaneous, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, patches, and the like. The most suitable route in any given case will depend on the severity of the condition being treated. The most preferred route of administration of the present invention is the oral route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In practical use, the compounds of formula (I) can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (e.g. intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used in the case of oral solid preparations such as, for example, powders, capsules, and tablets, with the solid oral preparations being preferred over the liquid preparations. The most preferred solid oral preparation is tablets.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques.

In addition to the common dosage forms set out above, the compounds of formula (I) may also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200; 4,008,719; 4,687,660; and 4,769,027, the disclosures of which are hereby incorporated by reference.

Pharmaceutical compositions employed in the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, or aerosol sprays each containing a predetermined amount of the active ingredient as a powder or granules, a solution or a suspension in an aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

For example, a tablet may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The invention is further defined by reference to the following examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practised without departing from the purpose and interest of this invention.

EXAMPLES

Synthetic Examples

The invention is illustrated with reference to the following Examples, as set out in Table 1. The synthesis of these compounds is performed using the general Synthetic Methods A to Q described hereinafter. The Synthetic Method used for the preparation of each Example is given in parentheses in column 1 of Table 1. Analytical data are given in Table 2.

TABLE 1 Example Structure Compound Name 1(B)

6-chloro-4-(2-furyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine 2(N)

4-(2-furyl)-N,N-dimethyl-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 3(O)

4-(2-furyl)-N-(2-hydroxyethyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 4(B)

tert-butyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate 5(C)

4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 6(P)

6-amino-N-benzyl-4-(2-furyl-1H-pyrazolo[3,4-d]pyrimidine-1-carboxamide 7(D)

1-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 8(D)

1-(3-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 9(D)

4-(2-furyl)-1-(4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 10(D)

1-(3-cyanobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 11(D)

1-(2-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 12(D)

4-(2-furyl)-1-(3-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 13(D)

4-(2-furyl)-1,N,N-tris(3-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 14(D)

1-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 15(D)

4-(2-furyl)-1-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 16(D)

methyl 3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylmethyl)benzoate 17(D)

4-(2-furyl)-1-(4-sulphonylbenzyl)-1H-pyrazolo[3,4-d]pyrimdine-6-amine 18(D)

4-(2-furyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 19(D)

1-cyclohexylmethyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 20(G)

3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylmethyl)benzoic acid 21(D)

4-(2-furyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 22(D)

4-(2-furyl)-1-(4-trifluoromethylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 23(D)

4-(2-furyl)-1-n-pentyl-1H-pyrazolo[3,4-d]pyrimidine-6-amine 24(D)

1-(2,6-difluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3.4-d]pyrimidine-6-amine 25(D)

4-(2-furyl)-1-(3-pyridylmethyl)-1H-pylazolo[3,4-d]pyrimidine-6-amine 26(D)

ethyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetate 27(G)

6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetic acid 28(I)

1-(2-fluorobenzyl)-4-(2-thienyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 29(H)

6-amino-4-(2-furyl)-N-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 30(D)

4-(2-fury1)-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 31(D)

4-(2-furyl)-1-(3-phenylpropyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 32(E)

1-(3-aminobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 33(J)

1-(2-fluorobenzyl)-4-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 34(H)

6-amino-N-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 35(H)

6-amino-4-(2-furyl)-N-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 36(H)

6-amino-N-(3-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 37(H)

6-amino-4-(2-furyl)-N-(2-furylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 38(J)

1-(2-fluorobenzyl)-4-(2-thiazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 39(D)

4-(2-furyl)-1-(2-furylmethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 40(F)

4-(2-furyl)-1-(4-methylphenylsulphonyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 41(J)

1-(2-fluorobenzyl)-4-(1-methyl-2-imidazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 42(K)

1-(2-fluorobenzyl)-4-(2-imidazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 43(H)

6-amino-4-(2-furyl)-N-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 44(H)

6-amino-4-(2-furyl)-N-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 45(H)

6-amino-N-(2-fluorophenyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 46(D)

3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylmethyl)-N-methylbenzamide 47(D)

3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylmethyl)-N,N-dimethylbenzamide 48(H)

6-amino-4-(2-furyl)-N-(2-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 49(H)

6-amino-N-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide 50(K)

1-(4-methylbenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidne-6-amine 51(K)

1-(2-fluorobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 52(D)

4-(2-furyl)-1-(2-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 53(F)

N-(4-(2-furyl)-1-(3-(2-thiophenesulphonamido))benzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-yl)-2-thiophenesulphonamide 54(F)

N-((3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)phenyl-2-thiophenesulphonamide 55(K)

1-(2-fluorobenzyl)-4-(3-pyrazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 56(E)

1-(2-aminobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 57(F)

N-((3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)phenyl-1-methyl-1H-imidazol-4-sulphonamide 58(K)

1-(3-nitrobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 59(K)

1-(3-nitrobenzyl)-4-(3-pyrazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 60(E)

1-(3-aminobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 61(J)

1-(3-nitrobenzyl)-4-(2-thiazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 62(E)

1-(3-aminobenzyl)-4-(2-thiazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 63(E)

1-(3-aminobenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 64(K)

1-(3-methoxybenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimdine-6-amine 65(Q)

4-(2-furyl)-1-(4-indolylmethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine 66(D)

tert-butyl 4-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)indole-1-carboxylate Synthetic Methods Method A tert-Butyl 6-amino-4-chloro-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate

A solution of 2-amino-4-chloro-1H-pyrazolo[3,4-d]pyrimidine (848 mg, 5 mmol) in DMF (5 mL) was treated with Et₃N (834 μL, 6 mmol), di-t-butyl dicarbonate (1.31 g, 6 mmol) and a catalytic amount of DMAP, stirred at room temperature for 1 h, poured into water, extracted with EtOAc, dried (MgSO₄), concentrated in vacuo and purified by chromatography (EtOAc:Heptane, 1:1) to give the title compound (312 mg, 23%) as a cream solid.

Method B

tert-Butyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate (Example 4)

A solution of tert-butyl 6-amino-4-chloro-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate (269 mg, 1 mmol) in DMF (2 mL) was treated with PdCl₂(PPh₃)₂ (35 mg, 0.05 mmol) and 2-(tributylstannyl)furan (315 μL, 1 mmol), stirred at room temperature for 16 h and the resulting solid filtered and washed with EtOAc to give the title compound (298 mg, 99%) as a cream solid.

Method C

4-(2-Furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 5)

A solution of tert-butyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate (204 mg, 0.68 mmol) in MeOH (2 mL) was treated with dimethylamine in water (0.5 mL, 40%), refluxed for 1 h, cooled and the resulting solid filtered and washed with water to give the title compound (95 mg, 70%) as a cream solid.

Method D

1-(2-Fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 14)

A solution of 4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (219 mg, 1 mmol) in DMF (2 mL) at 0° C. was treated with NaH (40 mg, 60%, 1 mmol), stirred for 20 min, treated with 2-fluorobenzyl bromide (120 μL, 1 mmol), stirred at room temperature for 1 h, quenched with water, extracted with EtOAc, dried (MgSO₄), concentrated in vacuo and purified by chromatography EtOAc:Heptane, 1:4) to give the title compound (250 mg, 76%) as a cream solid.

4-(2-Furyl)-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 30)

A solution of 4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (603 mg, 3 mmol) in DMF (3 mL) at 0° C. was treated with NaH (120 mg, 60%, 3 mmol), stirred for 20 min, treated with 3-nitrobenzyl bromide (648 mg, 3 mmol), stirred at room temperature for 1 h, poured into water, extracted with EtOAc, dried (MgSO₄) and concentrated in vacuo. The crude product was purified by chromatography EtOAc:Heptane, 1:1) to give the title compound as a cream solid (472 mg, 47%). A portion of the solid (70 mg) was suspended in MeOH, treated with HCl in dioxan (4-M, 1 mL), diluted with diethyl ether and filtered to give the hydrochloride salt of the title compound (79 mg, 100%) as a yellow solid.

4-Chloro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine

This was prepared from 4-chloro-1H-pyrazolo[3,4-d]pyrimidine-6-amine by Method D to give the title compound (562 mg, 34%) as a cream solid: mp 197–198° C.; NMR δ_(H) (400 MHz, CDCl₃) 5.23 (1H, br s), 5.51 (2H, s), 7.03–7.16 (3H, m), 7.23–7.31 (1H, m) and 7.91 (1H, s); IR ν_(max) (Nujol)/cm⁻¹ 3489, 3288, 3169, 2923, 1631, 1454, 1378, 1223 and 760; Retention time: 2.60 min.

4-Chloro-1-(3-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine

This was prepared from 4-chloro-1H-pyrazolo[3,4-d]pyrimidine-6-amine by Method D to give the title compound: IR ν_(max) (DR)/cm⁻¹ 3435, 3321, 3217, 2923, 1603, 1456, 1378, 1254, 1155, 1034, 980 and 788; NMR δ_(H) (400 MHz, DMSO) 8.03 (1H, s), 7.32 (2H, br s), 7.22 (1H, t, J 7.5 Hz), 6.84 (1H, dd, J 9.0, 3.5 Hz), 6.79–6.75 (1H, m), 6.71 (1H, d, J 8.5 Hz), 5.36 (2H, s) and 3.71 (3H, s).

Method E

1-(3-Aminobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 32)

A suspension of 4-(2-furyl)-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (396 mg, 1.18 mmol) in EtOH (5 mL) was heated to 50° C., treated with a solution of SnCl₂.2H₂O (798 mg, 3.54 mmol) in conc. HCl (1.8 mL), stirred at 50° C. for 2 h then heated at 70° C. for 2 h. The reaction mixture was cooled, basified (5-M NaOH) filtered and the resulting solid suspended in MeOH, treated with HCl in dioxan (4-M, 2 mL), diluted with diethyl ether and filtered to give the title compound (419 mg, 94%) as a yellow solid.

Method F

N-((3-(6-Amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)phenyl-2-thiophenesulphonamide (Example 54)

A solution of 1-(3-aminobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (153 mg, 0.5 mmol) in pyridine (2 mL) was treated with 2-thiophenesulphonyl chloride (91 mg, 0.5 mmol), stirred at room temperature for 16 h, poured into water, extracted with EtOAc, dried (MgSO₄), concentrated in vacuo and purified by chromatography (MeOH:DCM, 1:99) to give the title compound (49 mg, 22%) as a cream solid.

Method G

6-Amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetic acid (Example 27)

A solution of ethyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetate (350 mg, 1.22 mmol) in MeOH (2 mL) was treated with NaOH (1-M, 1 mL, 1 mmol), refluxed for 15 min, cooled, acidified and filtered to give the title compound (300 mg, 95%) as a white solid.

Method H

6-Amino-4-(2-furyl)-N-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide (Example 29)

A solution of 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetic acid (200 mg, 0.77 mmol) in DMF (3 mL) was treated with carbonyl diimidazole (125 mg, 0.77 mmol), stirred at room temperature for 2 h, treated with 2-aminopyridine (72 mg, 0.77 mmol), heated to 50° C. for 3 h, cooled and diluted with water. The resulting white solid was filtered, suspended in MeOH, treated with HCl in dioxan (4-M, 1 mL) diluted with diethyl ether and filtered to give the title compound (187 mg, 59%) as a yellow solid.

Method I

1-(2-Fluorobenzyl)-4-(2-thienyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 28)

A stirred solution of 4-chloro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine(139 mg, 0.5 mmol) in dry THF (10 mL) was treated with 2-thiopheneboronic acid (128 mg, 1.0 mmol), Pd(PPh₃)₄ (50 mg, 10 mol %) and saturated aqueous NaHCO₃ solution (2 mL), refluxed for 30 min, diluted with H₂O, extracted with EtOAc, dried MgSO₄) and concentrated in vacuo. The resulting dark brown oil was purified by chromatography [SiO₂; heptane:EtOAc (1:1) then (1:3)] to give the title compound (32 mg, 20%) as a cream solid.

Method J

1-(2-Fluorobenzyl)-4-(2-thiazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 38)

A stirred solution of thiazole (0.14 mL, 2 mmol) in dry THF (10 mL) at −78° C., under argon was treated n-BuLi (1.6-M in hexanes, 1.25 mL, 2 mmol), stirred for 15 min, treated with ZnCl₂ solution (1-M in Et₂O, 2 mL, 2 mmol) then allowed to warm gradually to room temperature. The mixture was treated with 4-chloro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (278 mg, 1 mmol) and Pd(PPh₃)₄ (100 mg, 10 mol %), refluxed for 2 h, diluted with saturated NH₄Cl solution, extracted with EtOAc, dried (MgSO₄) and concentrated in vacuo. The resulting brown oil was purified by chromatography [SiO₂; heptane:EtOAc (4:1) then (3:1)] to give the title compund (101 mg, 31%) as a cream solid.

Method K

1-(2-Fluorobenzyl)-4-(3-pyrazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 55)

A stirred solution of 1-(2-trimethylsilylethyl)pyrazole (615 mg, 3.1 mmol) in dry THF (10 mL) at −78° C., under argon was treated with n-BuLi (1.6-M in hexanes, 1.9 mL, 3.1 mmol), stirred for 20 min, treated with ZnCl₂ solution (1-M in Et₂O, 3.1 mL, 3.1 mmol) and allowed to warm gradually to room temperature. The mixture was treated with 4-chloro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (430 mg, 1.55 mmol) and Pd(PPh₃)₄ (170 mg, 10 mol %), refluxed for 1 h, diluted with saturated NH₄Cl solution, extracted with EtOAc, dried (MgSO₄) and concentrated in vacuo. The resulting brown oil was purified by chromatography [SiO₂; heptane-EtOAc (3:1)] to give the coupled product as a cream solid. This material was dissolved in MeOH (10 mL), treated with anhydrous HCl (4-M in dioxan, 5 mL), stirred for 17 h, concentrated in vacuo, diluted with Et₂O and filtered to give the title compound (216 mg, 40%) as a cream solid.

Method L

5-Amino-1-(2-phenylethyl)-1H-pyrazole-4-carboxylic acid

A mixture of conc. H₂SO₄ (15 mL) and water (15 mL) was treated with 5-amino-1-(2-phenylethyl)-1H-pyrazole-4-carbonitrile (5.0 g, 23.6 mmol), heated at 60° C. for 1 h, poured into water, extracted with EtOAc, dried (MgSO₄), concentrated in vacuo, and recrystallised (EtOAc/MeOH) to give the title compound (2.53 g, 47%) as a cream solid.

Method M

6-Hydroxy-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol

A mixture of 5-amino-1-(2-phenylethyl)-1H-pyrazole-4-carboxylic acid (1.87 g, 8.1 mmol) and urea (1.46 g, 24 mmol) was heated at 180° C. for 5 h, cooled and the resulting solid suspended in boiling water, filtered and washed with water to give the impure title compound (2.18 g, 105%,) as a cream solid.

Method N

4,6-Dichloro-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine

A solution of 6-hydroxy-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol (2.13 g, 8.32 mmol) in PhPOCl₂ (8 mL) was heated to 165° C. for 16 h, cooled, poured onto ice and the solid isolated by filtration. The solid was suspended in DCM, filtered through a silica pad and the filtrate was concentrated in vacuo to give the title compound (556 mg, 23%) as a colourless oil.

Method O

4-(2-Furyl)-N-(2-hydroxyethyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 3)

A solution of 6-chloro-4-(2-furyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine (162 mg, 0.5 mmol) in NMP (1 mL) was treated with ethanolamine (60 μL, 1 mmol), heated at 100° C. for 16 h, cooled and purified by chromatography (EtOAc:Heptane, 2:1). The resulting colourless oil was crystallised (MeOH/water) and filtered to give the title compound (100 mg, 57%) as a white solid.

Method P

6-Amino-N-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxamide (Example 6)

A solution of 4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (210 mg, 1 mmol) in DMF (2 mL) was treated with benzyl isocyanate (123 μL, 1 mmol) and a catalytic amount of DMAP, stirred at room temperature for 1 h, poured into water, extracted with EtOAc, dried (MgSO₄), concentrated in vacuo and purified by preparative HPLC (MeOH/H₂O gradient) to give the title compound (80 mg, 24%) as a pale yellow solid. Method Q 4-(2-Furyl)-1-(4-indolymethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (Example 65)

A solution of tert-butyl 4-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)indole-1-carboxylate (92 mg, 0.17 mmol) in MeOH (3 mL) was treated with NaOMe (45 mg, 0.84 mmol), refluxed for 2 h, cooled, diluted with water (15 mL) and filtered to give the title compound (45 mg, 82%) as an off white powder.

TABLE 2 HPLC is carried out using the following conditions: Column, Supelcosil ABZ⁺ (170 × 4.6 mm), particle size 5 μM, mobile phase MeOH: 10 mM aq NH₄OAc (80:50), flow rate 1.0 mL/min., detection wavelength λ 230 nM. Retention times ae provided in Table 2 Example Yield(%) Physical Data 1 96 Mp85.1–85.6° C.; IRν_(max) Nujol)/cm⁻¹ 2925, 2854, 1604, 1562, 1465, 1377, 1334, 1148, 1014 and 926; NMR(CDCl₃)8.48(1H, s), 7.80(1H, d, J1.2Hz), 7.60(1H, d, J3.6Hz), 7.27–7.17(5H, m), 6.69(1H, dd, J 3.6, 2.0Hz), 4.69(2H, m) and 3.26(2H, t, J8.0Hz); Anal. Calcd for C₁₇H₁₃ClN₄O: C, 67.89; H, 4.00; N, 17.24. Found: C, 62.70; H, 4.06; N, 16.99. 2 67 Mp90.9–91.1° C.; IRν_(max) Nujol)/cm⁻¹ 2926, 2855, 1609, 1584, 1549, 1488, 1456, 1401, 1160, and 1010; NMR(CDCl₃)8.21(1H, s), 7.71(1H, d, J1.0Hz), 7.36(1H, d, J3.3Hz), 7.28–7.17(5H, m), 6.61(1H, dd, J 3.4, 1.8Hz), 4.53(2H, m), 3.28(6H, s) and 3.23(2H, t, J7.8Hz); Anal. Calcd for C₁₉H₁₉N₅O: C, 68.45; H, 5.74; N, 21.00. Found: C, 68.40; H, 5.77; N, 20.90. 3 57 Mp118.6–119.3° C.; IRν_(max) Nujol)/cm⁻¹ 3317, 3099, 1613, 1568, 1543 and 1481; NMR δH(400MHz, CDCl₃)3.21(2H, t, J7.5Hz), 3.60–3.68 (2H, m), 3.83–3.89(2H, m), 4.51(2H, t, J7.5Hz), 5.61(1H, s), 6.59–6.63 (1H, m), 7.15–7.28(5H, m), 7.32–7.37(1H, m), 7.69–7.74(1H, m)and 8.22(1H, s); Anal. Calcd for C₁₉H₁₉N₅O₂: C, 65.32; H, 5.48; N, 20.03. Found: C, 65.24; H, 5.48; N, 19.91. 4 99 Mp261.9–262.7° C.; NMR δ_(H) 400MHz, DMSO)1.61(9H, s), 6.79–6.82 (1H, m), 7.18(2H, s), 7.44(1H, d, J3.0Hz), 8.08(1H, s), 8.42(1H, s); Anal. Calcd for C₁₄H₁₅N₅O₃ · 0.4 H₂O: C, 54.50; H, 5.16; N, 22.70. Found: C, 54.84; H, 5.18; N, 22.35. 5 70 Mp237.5–238.2° C.; IRν_(max) Nujol)/cm⁻¹ 3498, 3341, 3210, 3134, 2924, 2854, 1663, 1639, 1596, 1561, 1463 and 1377; NMR δ_(H) 400MHz, DMSO)6.70(2H, s), 6.76–6.78(1H, m), 7.40(1H, d, J3.5Hz), 8.04(1H, s), 8.21(1H, s)and 12.99(1H, s). 6 24 Mp171.2–171.6° C.; IRν_(max) Nujol)/cm⁻¹ 3461, 3307, 3200, 3063, 3029, 1643, 1613, 1576, 1557, 1479 and 1379; NMR δ_(H) 400MHz, DMSO)4.56(2H, d, J6.0Hz), 6.81–6.83(1H, m), 7.23–7.30(1H, m), 7.30–7.41(5H, m), 7.49(1H, d, J3.5Hz), 8.10(1H, s), 8.47(1H, s)and 9.30(1H, t, J6.2Hz); Anal. Calcd for C₁₇H₁₄N₆O₂ · 0.5 H₂O: C, 59.47; H, 4.40; N, 24.48. Found: C, 59.75; H, 4.22; N, 24.45. 7 Mp189.6–189.8° C.; IRν_(max) Nujol)/cm⁻¹ 3324, 3195, 2955, 2854, 1655, 1580, 1563, 1514, 1483, 1446 and 1432; NMR δ_(H) 400MHz, DMSO) 8.27(1H, s), 8.06(1H, d, J1.2Hz), 7.44(1H, d, J3.6Hz), 7.33–7.18 (3H, m), 6.91(2H, br s), 6.79(1H, dd, J3.2, 1.6Hz)and 5.43(2H, s); Anal Calcd for C₁₆H₁₀N₅O · 0.4 H₂O: C, 64.38; H, 4.66; N, 23.46. Found: C, 64.58; H, 4.51; N, 23.18. 8 37 Mp187.7–187.8° C.; IRν_(max) Nujol)/cm⁻¹ 3363, 3323, 3201, 2924, 2854, 1645, 1610, 1580, 1557, 1514, 1483 and 1445; NMR δ_(H) 400MHz, DMSO)8.30(1H, s), 8.06(1H, d, J0.8Hz), 7.45(1H, d, J3.2Hz), 7.38–7.13(3H, m), 6.94(2H, br s), 6.79(1H, dd, J3.2, 1.6Hz)and 5.44(2H, s); Anal. Calcd for C₁₆H₁₂ClN₅O · 0.1 H₂O: C, 58.67; H, 3.75; N, 21.38. Found: C, 58.97; H, 3.86; N, 20.95. 9 Mp240.3–240.8° C.; IRν_(max) Nujol)/cm⁻¹ 3325, 3204, 2922, 2854, 1652, 1605, 1580, 1555, 1523, 1467, 1377 and 1344; NMR δ_(H) 400MHz, DMSO)8.28(1H, s), 8.16(2H, d, J8.8Hz), 8.01(1H, s), 7.45–7.42 (3H, m), 6.76(1H, m), 6.70(2H, br s), and 5.57(2H, s); Anal. Calcd for C₁₆H₁₂N₆O₃ · 0.7 H₂O: C, 49.87; H, 3.77; N, 21.81. Found: C, 50.09; H, 3.46; N, 21.59. 10 Mp226.8–226.9° C.; NMR δ_(H) 400MHz, DMSO)8.31(1H, s), 8.06 (1H, d, J1.2Hz), 7.75(1H, d, J7.6Hz), 7.67(1H, s), 7.57–7.45(3H, m), 6.95(2H, br s), 6.79(1H, dd, J3.6, 1.6Hz)and 5.50(2H, s); Anal. Calcd for C₁₇H₁₂N₆O · 0.75 H₂O: C, 61.91; H, 4.13; N, 25.48. Found: C, 62.14; H, 3.81; N, 25.37. 11 Mp159.4–162.2° C.; NMR δ_(H) 400MHz, DMSO)8.32(1H, s), 8.07 (1H, s), 7.49(1H, d, J8.0Hz), 7.46(1H, d, J3.4Hz), 7.34–7.24(2H, m), 6.94(2H, br s), 6.80(1H, dd, J3.6, 1.9Hz)and 5.50(2H, s). 12 Mp191.7–191.8° C.; NMR δ_(H) 400MHz, DMSO)8.28(1H, s), 8.05 (1H, d, J0.8Hz), 7.44(1H, d, J3.6Hz), 7.22(1H, t, J8.0Hz), 6.91(2H, s), 6.85–6.72(5H, m), 5.93(2H, s)and 3.70(3H, s). Anal. Calcd. for C₁₇H₁₅N₅O₂ · 0.2 H₂O: C, 62.84; H, 4.78; N, 21.55. Found: C, 62.91; H, 4.71; N, 21.22. 13 IRν_(max) Nujol)/cm⁻¹ 2923, 2854, 1616, 1588, 1552, 1531, 1487, 1456, 1377, 1245 and 1042; NMR δ_(H) 400MHz, DMSO)8.30(1H, s), 8.07 (1H, s), 7.45(1H, d, J3.6Hz), 7.20–7.11(3H, m), 6.92–6.71(10H, m), 5.38(2H, s), 4.98–4.89(4H, m)and 3.66(9H, s); Anal. Calcd for C₃₃H₂₁N₅O₄ · H₂O: C, 68.38; H, 5.74; N, 12.08; Found: C, 68.74; H, 5.47; N, 12.10; M/Z 562(M + H)⁺. 14 28 Mp210.3–210.4° C.; IRν_(max) Nujol)/cm⁻¹ 3339, 3203, 3061, 1655, 1582, 1561, 1407, 1454, 1228 and 758; NMR δ_(H) 400MHz, DMSO) 5.47(2H, s), 6.77–6.80(1H, m), 6.93(2H, s), 7.04–7.09(1H, m), 7.10–7.16 (1H, m), 7.19–7.26(1H, m), 7.31–7.38(1H, m), 7.44(1H, d, J 3.0Hz), 8.06(1H, s)and 8.28(1H, s). 15 Mp226.9–227.7° C.; IRν_(max) Nujol)/cm⁻¹ 3331, 3111, 2925, 2854, 1638, 1574, 1488, 1460, 1378, 1032 and 887; NMR δ_(H) 400MHz, DMSO)8.40(1H, s), 8.17(1H, s), 7.75(1H, s), 7.21(1H, t, J7.2Hz), 7.10–7.00(3H, m), 6.88(1H, dd, J3.6, 1.6Hz)and 5.39(2H, s); Anal. Calcd for C₁₇H₁₅N₅O · HCl · H₂O: C, 56.75; H, 5.04; N, 19.46. Found: C, 57.03; H, 5.06; N, 19.10. 16 40 Mp202.3–202.4° C.; IRν_(max) Nujol)/cm⁻¹ 3408, 3327, 3212, 2924, 2854, 1724, 1640, 1607, 1579, 1558, 1510, 1485 and 1448; NMR δ_(H) (400MHz, DMSO)8.30(1H, s), 8.06(1H, d, J1.2Hz), 7.87(1H, m), 7.81(1H, s), 7.52–7.45(3H, m), 6.94(2H, s), 6.79(1H, dd, J3.6, 2.0Hz), 5.51(2H, s)and 3.83(3H, s). 17 Mp239.2–239.4° C.; IRν_(max) Nujol)/cm⁻¹ 3318, 3136, 2922, 2854, 1656, 1634, 1614, 1586, 1463, 1377, 1300 and 1148; NMR δ_(H) 400MHz, DMSO)8.37(1H, s), 8.11(1H, s), 7.88(2H, d, J8.4Hz), 7.58 (1H, d, J3.4Hz), 7.42(2H, d, J8.3Hz), 6.83(1H, dd, J3.6, 1.7Hz), 5.6 (2H, s)and 3.18(3H, s); Anal. Calcd for C₁₇H₁₅N₅O₃S · HCl · 0.5 H₂O: C, 49.22; H, 4.13; N, 16.88. Found: C, 49.05; H, 3.93; N, 16.53. 18 NMR δ_(H) 400MHz, DMSO)8.37(1H, s), 8.16(1H, s), 7.73(1H, s), 7.27–7.16 (5H, m), 6.87(1H, dd, J3.4, 1.6Hz), 4.44(2H, d, J7.1Hz)and 3.16(2H, t, J7.2Hz); Anal. Calcd for C₁₇H₁₅N₅O · HCl: C, 59.74; H, 4.72; N, 20.48. Found: C, 59.46; H, 4.76; N, 20.11. 19 NMR δ_(H) 400MHz, DMSO)8.32(1H, s), 8.12(1H, d, J1.2Hz), 6.84 (1H, dd, J3.6, 2.0Hz), 4.04(2H, d, J7.6Hz), 1.92(1H, m), 1.65–1.50 (5H, m)and 1.16–1.14(5H, m); Retention time 5.59 min. 20 99 Mp306.0–306.3° C.; NMR δ_(H) 400MHz, DMSO)8.32(1H, s), 8.08 (1H, s), 7.85(1H, m), 7.77(1H, s), 7.48(3H, m), 6.81(1H, dd, J3.6, 1.6Hz), 7.0(2H, br s)and 5.50(2H, s); Retention time 0.62 min. 21 Mp191.4–191.5° C.; IRν_(max) Nujol)/cm⁻¹ 3456, 3328, 3121, 2924, 2854, 1662, 1633, 1573, 1484, 1454, 1375 and 1032; NMR δ_(H) 400MHz, DMSO)8.33(1H, s), 8.14(1H, s)7.66(1H, br s), 6.86(1H, br s), 4.89(1H, br m)and 1.45(6H, br m); Anal. Calcd for C₁₂H₁₃N₅O · HCl · 1.25 H₂O: C, 47.69; H, 5.50; N, 23.17. Found: C, 47.90; H, 5.21; N, 23.00. 22 Mp219.0–219.4° C.; IRν_(max) Nujol)/cm⁻¹ 2924, 2854, 1644, 1574, 1461, 1377, 1328 and 1112; NMR δ_(H) 400MHz, DMSO)8.35(1H, s), 8.10(1H, s), 7.69(2H, d, J8.0Hz), 7.55(1H, s), 7.39(2H, d, J8.0Hz), 6.82(1H, t, J1.6Hz)and 5.54(2H, s); Anal. Calcd for C₁₇H₁₂F₃N₅O · HCl · 0.5 H₂O: C, 50.44; H, 3.49; N, 17.30. Found: C, 50.73; H, 3.23; N, 17.19. 23 Mp218° C.; NMR δ_(H) 400MHz, DMSO)8.36(1H, s), 8.16(1H, s), 7.73 (1H, s), 6.87(1H, t, J1.8Hz), 4.19(2H, m), 7.80(2H, m), 1.32–1.23 (4H, m)and 0.84(3H, t, J6.8Hz); Anal. Calcd for C₁₄H₁₇N₅O · HCl: C, 54.63; H, 5.89; N, 22.74. Found: C, 54.55; H, 6.04; N, 22.44. 24 Mp235.5–236.2° C.; NMR δ_(H) 400MHz, DMSO)8.20(1H, s), 8.12 (1H, s), 7.61(1H, d, J3.2Hz), 7.50–7.43(1H, m), 7.14–7.11(2H, m), 6.83(1H, dd, J3.6, 1.6Hz)and 5.44(2H, s); Anal. Calcd for C₁₆H₁₁F₂N₅O · HCl · 0.75 H₂O: C, 50.94; H, 3.61; N, 18.56. Found: C, 50.96; H, 3.32; N, 18.20. 25 10 NMR δ_(H) 400MHz, DMSO)5.61(2H, s), 6.79–6.83(1H, m), 7.52(1H, d, J3.5Hz), 7.82(1H, dd, J8.0, 5.5Hz), 8.09(1H, s), 8.12(1H, s), 8.76 (1H, s); M/Z 293(M + H)⁺. 26 41 Mp177.0–177.1° C.; IRν_(max) Nujol)/cm⁻¹ 3456, 3331, 3211, 3142, 3121, 2726, 1731, 1651, 1587, 1559, 1488, 1462, 1377, 1235 and 755; NMR δ_(H) 400MHz, DMSO)1.20(3H, t, J7.0Hz), 4.15(2H, q, J7.0Hz) 5.06(2H, s), 6.78–6.81(1H, m), 6.94(2H, s), 7.44(1H, d, J3.5Hz), 8.06(1H, s)and 8.28(1H, s); Anal. Calcd for C₁₃H₁₃N₅O₃: C, 54.35; H, 4.56, N, 24.37. Found: C, 54.22; H, 4.58; N, 24.01. 27 95 Mp291.9–192.0° C.; IRν_(max) Nujol)/cm⁻¹ 3371, 3329, 3204, 3141, 2726, 1704, 1658, 1579, 1456, 1378, 956 and 789; NMR δ_(H) 400MHz, DMSO)4.96(2H, s), 6.78–6.80(1H, m), 6.92(2H, s), 7.44(1H, d, J3.5Hz), 8.05–8.07(1H, m), 8.26(1H, s)and 13.09(1H, s); Anal. Calcd for C₁₁H₉N₅O₃ · 0.3 H₂O: C, 49.93; H, 3.66, N, 26.46. Found: C, 50.22; H, 3.46; N, 26.12. 28 20 Mp204.5° C.; IRν_(max) Nujol)/cm⁻¹ 3485, 3322, 2925, 1651, 1587, 1378, 1232 and 709; NMR δ_(H) 400MHz, DMSO)5.14(2H, br s), 5.57(2H, s), 7.01–7.14(3H, m), 7.20–7.31(2H, m), 7.59(1H, dd, J5.0, 1.0Hz), 7.98(1H, d, J3.5Hz)and 8.19(1H, s); Retention time 4.87 min 29 59 Mp249.8–249.9° C.; IRν_(max) Nujol)/cm⁻¹ 3441, 3292, 3058, 2645, 1705, 1668, 1630, 1582, 1561, 1457, 1381 and 1215; NMR δ_(H) 400MHz, DMSO)5.25(2H, s), 6.16–6.78(2H, s), 6.91–6.96(1H, m), 7.23 (1H, t, J7.0Hz), 7.88–7.99(3H, m), 8.24(1H, s), 8.37(1H, d, J5.0Hz), 8.49(1H, s)and 11.41(1H, s); Anal. Calcd for C₁₆H₁₃N₇O₃ · HCl · 3 H₂O: C, 43.49; H, 4.56; N, 22.19. Found: C, 43.60; H, 4.01; N, 21.95. 30 47 IRν_(max) Nujol)/cm⁻¹ 3604, 3342, 3152, 3090, 2601, 1659, 1629, 1574, 1558, 1526 and 1460; NMR δ_(H) 400MHz, DMSO)4.66–5.19(2H, s), 5.59(2H, s), 6.82–6.85(1H, m), 7.60(1H, d, J3.5Hz), 7.62–7.67(2H, m), 8.07–8.09(1H, m), 8.11(1H, d, J1.0Hz), 8.14–8.17(1H, m), 8.39 (1H, s); Anal. Calcd for C₁₆H₁₂N₆O₃ · HCl · 0.75 H₂O: C, 49.75; H, 3.78, N, 21.76. Found: C, 49.92; H, 3.39; N, 21.33. 31 Mp190.9–191.7° C.; IRν_(max) Nujol)/cm⁻¹ 3407, 3171, 3089, 2923, 2854, 1651, 1633, 1578, 1485, 1455 and 1377; NMR δ_(H) 400MHz, DMSO)8.40(1H, s), 8.17(1H, s), 7.76(1H, s), 7.30–7.15(5H, m), 6.88 (1H, dd, J3.5, 1.6Hz), 4.23(2H, t, J7.0Hz), 2.60(2H, t, J7.3Hz)and 2.17–2.10(2H, m); Retention time 5.42 min. 32 94 NMR δ_(H) 400MHz, DMSO)4.70–5.44(2H, s), 5.49(2H, s), 6.86(1H, s), 7.12(1H, s), 7.27(2H, t, J7.5Hz), 7.45(1H, t, J7.5Hz), 7.66(1H, d, J2.5Hz), 8.39(1H, s)and 9.48–10.63(2H, s); M/Z 307(M + H)⁺. 33 19 IRν_(max) Nujol)/cm⁻¹ 3328, 3193, 2925, 2854, 1650, 1598, 1561, 1513, 1456, 1430 and 1377; NMR δ_(H) 400MHz, DMSO)8.84(1H, d, J4.4Hz), 8.54(1H, s), 8.43(1H, d J8.0Hz), 8.04(1H, m), 7.60(1H, dd, J 6.8, 5.2Hz), 7.34(1H, m), 7.22(1H, m), 7.13(1H, m)7.07(1H, m), 7.03 (2H, s)and 5.50(2H, s). 34 58 Mp276.8–277.2° C.; NMR δ_(H) 400MHz, DMSO)4.29(2H, d, J6.0Hz), 4.89(2H, s), 6.77–6.81(1H, m), 6.88(2H, s), 7.21–7.37(5H, m), 7.44(1H, d, J3.5Hz), 8.04–8.07(1H, m), 8.26(1H, s)and 8.50(1H, t, J6.0Hz). 35 58 Mp276.8–277.2° C.; NMR δ_(H) 400MHz, DMSO)4.29(2H, d, J6.0Hz), 4.89(2H, s), 6.77–6.81(1H, m), 6.88(2H, s), 7.21–7.37(5H, m), 7.44(1H, d, J3.5Hz), 8.04–8.07(1H, m), 8.26(1H, s)and 8.50(1H, t, J6.0Hz). 36 29 Mp279.9–281.0° C.; NMR δ_(H) 400MHz, DMSO)5.08(2H, s), 6.78–6.82 (1H, m), 6.90(2H, s), 7.13(1H, d), J8.0Hz), 7.36(1H, t, J8.0Hz), 7.42–7.49(2H, m), 7.74–7.79(1H, m), 8.05–8.09(1H, m), 8.28(1H, s)and 10.52(1H, s). 37 49 Mp247.9–249.4° C.; NMR δ_(H) 400MHz, DMSO)4.28(2H, d, J6.0Hz), 4.86(2H, s), 6.26(1H, d, J2.5Hz), 6.38–6.41(1H, m), 6.77–6.80 (1H, m), 6.88(2H, s), 7.44(1H, d, J3.5Hz), 7.57–7.60(1H, m), 8.04–8.07 (1H, m), 8.25(1H, s)and 8.52(1H, t, J5.5Hz). 38 31 Mp200.0–200.1° C.; IRν_(max) Nujol)/cm⁻¹ 3473, 3181, 3078, 2923, 1646, 1572, 1461, 1376, 792 and 768; NMR δ_(H) 400MHz, DMSO)5.19(2H, br s), 5.59(2H, s), 6.98–7.12(3H, m), 7.20–7.29(1H, m), 7.58(1H, d, J3.5Hz)and 8.59(1H, s); Retention time 4.64 min. 39 Mp223.2–225.1° C.; NMR δ_(H) 400MHz, DMSO)8.29(1H, s), 8.09 (1H, d, J1.2Hz), 7.58(1H, m), 7.53(1H, d, J3.2Hz), 6.81(1H, dd, J 3.6, 2.0Hz), 6.41(1H, dd, J3.2, 2.0Hz), 6.36(1H, d, J2.8Hz)and 5.40 (2H, s). 40 61 IRν_(max) Nujol)/cm⁻¹ 3495, 3321, 3210, 3118, 2727, 1635, 1604, 1579, 1478, 1464, 1390 and 1175; NMR δ_(H) 400MHz, DMSO)2.36(3H, s), 6.78–6.79(1H, m), 7.43–7.45(4H, m), 7.92(2H, d, J8.5Hz), 8.05(1H, s)and 8.52(1H, s). 41 Mp218.7–219.5° C.; IRν_(max) Nujol)/cm⁻¹ 3495, 3321, 2925, 2854, 1615, 1596, 1570, 1512, 1481, 1452, and 1377; NMR δ_(H) 400MHz, DMSO)8.34(1H, s), 7.46(1H, s), 7.34(1H, s), 7.21(2H, m), 7.13(1H, m), 7.05(1H, m), 6.97(2H, br s), 5.47(2H, s)and 4.20(3H, s). 42 11 Mp250.5–251.2° C.; IRν_(max) Nujol)/cm⁻¹ 3483, 3354, 2924, 2854, 1616, 1601, 1575, 1503, 1454, 1418 and 1377; NMR δ_(H) 400MHz, DMSO)13.08(1H, br s), 8.37(1H, s), 7.38–7.06(6H, m)6.72(2H, br s)and 5.48(2H, s). 43 59 Mp267.4–271.5° C.; IRν_(max) Nujol)/cm⁻¹ 3461, 3290, 3194, 1664, 1589, 1465, 1377, 1291, 1235, 1157, 1014 and 795; NMR δ_(H) 400MHz, DMSO)3.71(3H, s), 5.06(2H, s), 6.65(1H, dd, J8.0, 2.0Hz), 6.78–6.81 (1H, m), 6.89(2H, s), 7.09(1H, d, J9.0Hz), 7.21(1H, d, J3.0Hz), 7.26–7.30(1H, m), 7.45(1H, d, J3.0Hz), 8.05–8.08(1H, m), 8.28 (1H, s)and 10.31(1H, s). 44 71 Mp231.1–231.2° C.; IRν_(max) Nujol)/cm⁻¹ 3462, 3298, 3213, 3111, 1651, 1586, 1558, 1487, 1456, 1377, 1280, 1169, 1009 and 765; NMR δ_(H) (400MHz, DMSO)2.72(2H, t, J7.0Hz), 3.24–3.35(2H, m), 4.80(2H, s), 6.77–6.82(1H, m), 6.88(2H, s), 7.16–7.23(3H, m), 7.24–7.33 (2H, m), 7.44(1H, d, J3.0Hz), 8.02–8.09(2H, m)and 8.25(1H, s). 45 15 Mp276.2–278.5° C.; IRν_(max) Nujol)/cm⁻¹ 3448, 3288, 3199, 1669, 1588, 1557, 1456, 1378, 1309, 1106, 888 and 757; NMR δ_(H) 400MHz, DMSO)5.15(2H, s), 6.76–6.83(1H, m), 6.90(2H, s), 7.11–7.20(2H, m), 7.23–7.32(1H, m), 7.42–7.48(1H, m), 7.83–7.93(1H, m), 8.04–8.09 (1H, m), 8.28(1H, s)and 10.12(1H, s). 46 22 NMR δ_(H) 400MHz, DMSO)8.39(1H, m), 8.29(1H, s), 8.06(1H, d, J 1.2Hz), 7.72–7.68(2H, m), 7.45–7.31(2H, m), 6.92(2H, s), 6.79(1H, dd, J3.2, 1.6Hz), 5.47(2H, s)and 2.74(3H, d, J4.8Hz); Anal. Calcd for C₁₈H₁₆N₆O₂ · 0.5 H₂O: C, 60.50; H, 4.79; N, 23.52. Found: C, 60.32; H, 4.63; N, 23.50. 47 33 NMR δ_(H) 400MHz, DMSO)8.29(1H, s), 8.06(1H, s), 7.44(1H, d, J3.4Hz), 7.40–7.20(4H, m), 6.92(2H, s), 6.79(1H, dd, J3.5, 1.9Hz)5.46 (2H, s), 2.94(3H, br s)and 2.85(3H, br s). 48 75 NMR δ_(H) 400MHz, DMSO)3.79(3H, s), 4.25(2H, d, J6.0Hz), 4.90 (2H, s), 6.75–6.81(1H, m), 6.84–7.01(4H, m), 7.18–7.28(2H, m), 7.44(1H, d, J3.5Hz), 8.06(1H, s), 8.27(1H, s), 8.29(1H, t, J6.0Hz). Anal. Calcd. for C₁₉H₁₈N₆O₃ · 0.5 H₂O: C, 58.91; H, 4.94; N, 21.69. Found: C, 60.20; H, 5.34; N, 21.33; M/Z 379(M + H)⁺. 49 75 NMR δ_(H) 400MHz, DMSO)4.33(2H, d, J5.0Hz), 4.90(2H, s), 6.79 (1H, s), 6.89(2H, s), 7.11–7.23(2H, m), 7.26–7.40(2H, m), 7.44(1H, d, J3.0Hz), 8.06(1H, s), 8.27(1H, s)and 8.52(1H, t, J5.5Hz); M/Z 367(M + H)⁺. 50 89 Mp. 265.8° C.; IRν_(max) Nujol)/cm⁻¹ 3360, 3290, 3147, 2924, 2854, 1637, 1608, 1575, 1497, 1465 and 1377; NMR δ_(H) 400MHz, DMSO)13.8 (1H, br s), 8.45(1H, s), 8.02(1H, s), 7.22(1H, br s), 7.13(4H, s), 5.39 (2H, s)and 2.26(3H, s). 51 18 Mp. 287.9° C.; NMR δ_(H) 400MHz, DMSO)8.77(1H, br s), 8.40(1H, s), 7.37–7.11(4H, m)and 5.50(2H, s). 52 80 NMR δ_(H) 400MHz, DMSO)8.35(1H, s), 8.14(1H, dd, J8.1, 1.1Hz), 8.07(1H, d, J1.2Hz), 7.67–7.56(2H, m), 7.47(1H, d, J3.5Hz), 6.95 (2H, s), 6.81–6.79(2H, m)and(5.80(2H, s); Anal. Calcd for C₁₆H₁₂N₆O₃ · 0.3 H₂O: C, 56.24; H, 3.72; N, 24.59. Found: C, 56.41; H, 3.48; N, 24.15. 53 IRν_(max) Nujol)/cm⁻¹ 2924, 2854, 1605, 1581, 1465, 1377, 1156 and 1018; NMR δ_(H) 400MHz, DMSO)12.0(1H, br s), 10.39(1H, br s), 8.52 (1H, s), 8.16(1H, s), 7.81(2H, br s), 7.75(1H, d, J4.0Hz), 7.60(1H, s), 7.38(1H, d, J2.4Hz), 7.23(1H, t, J7.6Hz), 7.05–6.85(5H, m)and 5.49(2H, s); Anal. Calcd for C₂₀H₁₆N₆O₃S₂ · 0.4 H₂O: C, 47.57; H, 3.13; N, 13.87. Found: C, 47.70; H, 3.20; N, 13.53. 54 22 NMR δ_(H) 400MHz, DMSO)5.36(2H, s), 6.78–6.82(1H, m), 6.87(1H, s), 6.89–6.95(2H, m), 6.98–7.04(3H, m), 7.21(1H, t, J8.0Hz), 7.38–7.40 (1H, m), 7.46(1H, d, J3.5Hz), 7.81(1H, dd, J5.0, 1.0Hz), 8.07 (1H, s), 8.28(1H, s)and 10.39(1H, s); M/Z 453(M + H)⁺. 55 40 Mp245.3–246.1° C.; IRν_(max) Nujol)/cm⁻¹ 3666, 2922, 1638, 1460, 1378, 1229 and 757; NMR δ_(H) 400MHz, DMSO)5.49(2H, s), 7.12–7.28(3H, m), 7.31–7.42(2H, m), 8.06(1H, d, J2.0Hz)and 8.53(1H, s); Retention time 2.10 min. 56 IRν_(max) Nujol)/cm⁻¹ 3600–2500 br, 2921, 2853, 1659, 1633, 1574, 1556, 1462, 1378 and 1031; NMR δ_(H) 400MHz, DMSO)8.38(1H, s), 8.13(1H, d, J1.2Hz), 7.37–7.29(2H, m), 7.12(1H, m), 6.88–6.84 (2H, m)and 5.54(2H, s). 57 6 NMR δ_(H) 400MHz, DMSO)3.68(3H, s), 5.49(2H, s), 6.60(1H, d, J7.5Hz), 6.79–6.82(1H, m), 6.91(2H, s), 7.02–7.09(1H, m), 7.18(2H, d, J4.5Hz), 7.47(1H, d, J3.5Hz), 7.23(1H, s), 7.84(1H, s), 8.07(1H, s), 8.31(1H, s)and 10.13(1H, s); M/Z 451(M + H)⁺. 58 14 NMR δ_(H) 400MHz, DMSO)8.73(1H, br s), 8.40(1H, s), 8.15(1H, m), 8.08(1H, s), 7.64(1H, d, J5.1Hz)and 5.61(2H, s). 59 5 Mp278.5–279.0° C.; NMR δ_(H) 400MHz, DMSO)8.44(1H, s), 8.15 (1H, m), 8.08(1H, s), 7.97(1H, d, J2.3Hz), 7.10(1H, s), 7.10(2H, br s) and 6.00(2H, s). 60 22 Mp193.3–193.4° C.; IRν_(max) Nujol)/cm⁻¹ 3289, 2934, 1661, 1642, 1582, 1514, 1460 and 1377; NMR δ_(H) 400MHz, DMSO)8.76(1H, s), 8.40(1H, s), 7.44(1H, t, J7.6Hz), 7.25(2H, m)7.07(1H, s)and 5.50 (2H, s). 61 25 Mp193.3–193.4° C.; NMR δ_(H) 400MHz, DMSO)5.61(2H, s), 7.17 (2H, br s), 7.61–7.67(2H, m), 8.07–8.11(2H, m), 8.12–8.19(1H, m), 8.23(1H, d, J3.0Hz)and 8.43(1H, s); Retention time 4.70 min. 62 93 Mp177.8–178.3° C.; IRν_(max) Nujol)/cm⁻¹ 3302, 2925, 1634, 1462, 1377, 774 and 778; NMR δ_(H) 400MHz, DMSO)5.50(2H, s), 7.08(1H, s), 7.23–7.31(2H, m), 7.45(1H, t, J7.5Hz), 8.09(1H, d, J3.0Hz), 8.22 (1H, d, J3.0Hz)and 8.41(1H, s); Retention time 1.75 min. 63 67 IRν_(max) Nujol)/cm⁻¹ 3162, 2920, 1551, 1574, 1464, 1378, 1072 and 766; NMR δ_(H) 400MHz, DMSO)8.51(1H, s), 8.04(1H, d, J2.5Hz), 7.44 (1H, t, J8.0Hz), 7.31(1H, br s), 7.27–7.20(2H, m), 7.10(1H, s)and 5.49(2H, s). 64 81 Mp171–172° C.; IRν_(max) Nujol)/cm⁻¹ 3252, 2922, 1667, 1619, 1466, 1378, 1261, 1169, 769 and 751; NMR δ_(H) 400MHz, DMSO)8.59(1H, s), 8.10(1H, d, J2.5Hz), 7.51(1H, br s), 7.25(1H, t, J8.0Hz), 6.90–6.82 (2H, m), 6.82–6.77(1H, d, J8.0Hz), 5.43(2H, s)and 3.72(3H, s). 65 82 IRν_(max) Nujol)/cm⁻¹ 3318, 2923, 1640, 1455, 1455, 1378 and 750; NMR δ_(H) 400MHz, DMSO)11.13(1H, br s), 8.23(1H, s), 8.05(1H, d, J1.0Hz), 7.43(1H, d, J3.5Hz), 7.34–7.28(2H, m), 7.00(1H, t, J7.0Hz), 6.89(2H, br s), 6.78(1H, dd, J3.5, 2.0Hz), 6.73(1H, d, J6.5Hz), 6.53–6.49 (1H, m)and 5.63(2H, s). 66 NMR δ_(H) 400MHz, DMSO)8.25(1H, s), 8.06–8.04(1H, m), 7.99(1H, d, J8.5Hz), 7.68(1H, d, J3.5Hz), 7.43(1H, d, J3.5Hz), 7.26(1H, t, J 7.5Hz), 6.99(1H, d, J6.5Hz), 6.93(2H, br s), 6.86(1H, d, J4.0Hz), 6.79–6.77(1H, m), 5.65(2H, s)and 1.61(9H, s). Adenosine Receptor Binding Binding Affinities at hA_(2A) Receptors

The compounds were examined in an assay measuring in vitro binding to human adenosine A_(2A) receptors by determining the displacement of the adenosine A_(2A) receptor selective radioligand [³H]-CGS 21680 using standard techniques. The results are summarised in Table 3.

TABLE 3 Example K_(i) nM) Example 11 9 Example 12 2 Example 14 2 Example 15 2 Example 22 6 Example 24 2 Example 28 5 Example 30 4 Example 31 2 Example 32 4 Example 33 12 Example 51 71 Example 55 16 Example 56 8 Evaluation of Potential Anti-Parkinsonian Activity In Vivo Haloperidol-Induced Hypolocomotion Model

It has previously been demonstrated that adenosine antagonists, such as theophylline, can reverse the behavioural depressant effects of dopamine antagonists, such as haloperidol, in rodents (Mandhane S. N. et al., Adenosine A₂ receptors modulate haloperidol-induced catalepsy in rats. Eur. J. Pharmacol. 1997, 328, 135–141). This approach is also considered a valid method for screening drugs with potential antiparkinsonian effects. Thus, the ability of novel adenosine antagonists to block haloperidol-induced deficits in locomotor activity in mice can be used to assess both in vivo and potential antiparkinsonian efficacy.

Method

Female TO mice (25–30 g) obtained from TUCK, UK, are used for all experiments. Animals are housed in groups of 8 [cage size—40 (width)×40 (length)×20 (height)cm] under 12 hr light/dark cycle (lights on 08:00 hr), in a temperature (20±2° C.) and humidity (55±15%) controlled environment. Animals have free access to food and water, and are allowed at least 7 days to acclimatize after delivery before experimental use.

Drugs

Liquid injectable haloperidol (1 ml Serenance ampoules from Baker Norton, Harlow, Essex, each containing haloperidol BP 5 mg, batch # P424) are diluted to a final concentration of 0.02 mg/ml using saline. Test compounds are typically prepared as aqueous suspensions in 8% Tween. All compounds are administered intraperitoneally in a volume of 10 ml/kg.

Procedure

1.5 hours before testing, mice are administered 0.2 mg/kg haloperidol, a dose that reduces baseline locomotor activity by at least 50%. Test substances are typically administered 5–60 minutes prior to testing. The animals are then placed individually into clean, clear polycarbonate cages [20 (width)×40 (length)×20 (height) cm , with a flat perforated, Perspex lid]. Horizontal locomotor activity is determined by placing the cages within a frame containing a 3×6 array of photocells linked to a computer, which tabulates beam breaks. Mice are left undisturbed to explore for 1 hour, and the number of beams breaks made during this period serves as a record of locomotor activity which is compared with data for control animals for statistically significant differences.

6-OHDA Model

Parkinson's disease is a progressive neurodegenerative disorder characterised by symptoms of muscle rigidity, tremor, paucity of movement (hypokinesia), and postural instability. It has been established for some time that the primary deficit in PD is a loss of dopaminergic neurones in the substantia nigra which project to the striatum, and indeed a substantial proportion of striatal dopamine is lost (ca 80–85%) before symptoms are observed. The loss of striatal dopamine results in abnormal activity of the basal ganglia, a series of nuclei which regulate smooth and well co-ordinated movement (Blandini F. et al., Glutamate and Parkinson's Disease. Mol. Neurobiol. 1996, 12, 73–94). The neurochemical deficits seen in Parkinson's disease can be reproduced by local injection of the dopaminergic neurotoxin 6-hydroxydopamine into brain regions containing either the cell bodies or axonal fibres of the nigrostriatal neurones.

By unilaterally lesioning the nigrostriatal pathway on only one-side of the brain, a behavioural asymmetry in movement inhibition is observed. Although unilaterally-lesioned animals are still mobile and capable of self maintenance, the remaining dopamine-sensitive neurones on the lesioned side become supersenstive to stimulation. This is demonstrated by the observation that following systemic administration of dopamine agonists, such as apomorphine, animals show a pronounced rotation in a direction contralateral to the side of lesioning. The ability of compounds to induce contralateral rotations in 6-OHDA lesioned rats has proven to be a sensitive model to predict drug efficacy in the treatment of Parkinson's Disease.

Animals

Male Sprague-Dawley rats, obtained from Charles River, are used for all experiments. Animals are housed in groups of 5 under 12 hr light/dark cycle (lights on 08:00 hr), in a temperature (20±2° C.) and humidity (55±15%) controlled environment. Animals have free access to food and water, and are allowed at least 7 days to acclimatize after delivery before experimental use.

Drugs

Ascorbic acid, desipramine, 6-OHDA and apomorphine (Sigma-Aldrich, Poole, UK). 6-OHDA is freshly prepared as a solution in 0.2% ascorbate at a concentration of 4 mg/mL prior to surgery. Desipramine is dissolved in warm saline, and administered in a volume of 1 ml/kg. Apomorphine is dissolved in 0.02% ascorbate and administered in a volume of 2 mL/kg. Test compounds are suspended in 8% Tween and injected in a volume of 2 mL/kg.

Surgery

15 minutes prior to surgery, animals are given an intraperitoneal injection of the noradrenergic uptake inhibitor desipramine (25 mg/kg) to prevent damage to non-dopamine neurones. Animals are then placed in an anaesthetic chamber and anaesthetised using a mixture of oxygen and isoflurane. Once unconscious, the animals are transferred to a stereotaxic frame, where anaesthesia is maintained through a mask. The top of the animal's head is shaved and sterilised using an iodine solution. Once dry, a 2 cm long incision is made along the midline of the scalp and the skin retracted and clipped back to expose the skull. A small hole is then drilled through the skill above the injection site. In order to lesion the nigrostriatal pathway, the injection cannula is slowly lowered to position above the right medial forebrain bundle at −3.2 mm anterior posterior, −1.5 mm medial lateral from bregma, and to a depth of 7.2 mm below the duramater. 2 minutes after lowing the cannula, 2 μL of 6-OHDA is infused at a rate of 0.5 μL/min over 4 minutes, yeilding a final dose of 8 μg. The cannula is then left in place for a further 5 minutes to facilitate diffusion before being slowly withdrawn. The skin is then sutured shut using Ethicon W501 Mersilk, and the animal removed from the strereotaxic frame and returned to its homecage. The rats are allowed 2 weeks to recover from surgery before behavioural testing.

Apparatus

Rotational behaviour is measured using an eight station rotameter system provided by Med Associates, San Diego, USA. Each station is comprised of a stainless steel bowl (45 cm diameter×15 cm high) enclosed in a transparent Plexiglas cover running around the edge of the bowl, and extending to a height of 29 cm. To assess rotation, rats are placed in cloth jacket attached to a spring tether connected to optical rotameter positioned above the bowl, which assesses movement to the left or right either as partial (45°) or full (360°) rotations. All eight stations are interfaced to a computer that tabulated data.

Procedure

To reduce stress during drug testing, rats are initially habituated to the apparatus for 15 minutes on four consecutive days. On the test day, rats are given an intraperitoneal injection of test compound 30 minutes prior to testing. Immediately prior to testing, animals are given a subcutaneous injection of a subthreshold dose of apomorphine, then placed in the harness and the number of rotations recorded for one hour. The total number of full contralatral rotations during the hour test period serves as an index of antiparkinsonian drug efficacy. 

1. A compound of formula (I):

wherein: R₁ is selected from the group consisting of alkyl, alkoxy, aryloxy, alkylthio, arylthio, aryl, halogen, CN, NR₆R₇, NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈, and NR₅SO₂R₈; R₂ is a heteroaryl attached via an unsaturated ring carbon of said heteroaryl group, other than pyrazolopyrimidine; R₃ is selected from the group consisting of H, alkyl, halogen, OR₅, SR₅, and NR₆R₇; R₄ is selected from the group consisting of H, acyclic alkyl, CONR₆R₇, CONR₅NR₆R₇, COR₆, CO₂R₈, and SO₂R₈; R₅, R₆, and R₇ are independently selected from the group consisting of H, alkyl, and aryl, or where R₆ and R₇ are in an NR₆R₇ group, R₆ and R₇ may be linked to form a heterocyclic group, or where R₅, R₆ and R₇ are in a (CONR₅NR₆R₇) group, R₅ and R₆ may be linked to form a heterocyclic group; and R₈ is selected from alkyl and aryl, or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein R₁ is selected from the group consisting of alkyl, alkoxy, thioalkyl, NR₆R₇, NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈, and NR₅SO₂R₈.
 3. The compound of claim 1, wherein R₁ is selected from the group consisting of NR₆R₇, NR₅COR₆, NR₅CONR₆R₇, NR₅CO₂R₈, and NR₅SO2R₈.
 4. The compound of claim 1, wherein R₁ is NR₆R₇.
 5. The compound of claim 1, wherein R₁ is NH₂.
 6. The compound of claim 1, wherein R₁ is selected from the group consisting of NR₅CONR₆R₇, NR₅COR₆, NR₅CO₂R₈, and NR₅SO₂R₈, and R₅ is H or alkyl.
 7. The compound of claim 1, wherein R₁ is selected from the group consisting of NR₅CONR₆R₇, NR₅COR₆, NR₅CO₂R₈, and NR₅SO₂R₈, and R₅ is H.
 8. The compound of claim 1, wherein R₁ is selected from haloalkyl and arylalkyl.
 9. The compound of claim 1, wherein R₂ is a 5- or 6-membered monocyclic heteroaryl group.
 10. The compound of claim 1, wherein R₂ is a heteroaryl group which is attached to the pyrimidine ring of formula (I) such that a heteroatom is adjacent to said unsaturated carbon atom attached to the pyrimidine ring.
 11. The compound of claim 1, wherein R₂ is an N, O, or S-containing heteroaryl group.
 12. The compound of claim 1, wherein R₂ is unsubstituted in at least one ortho position.
 13. The compound of claim 1, wherein R₂ is unsubstituted at both ortho positions.
 14. The compound of claim 1, wherein R₂ is selected from the group consisting of furyl, thienyl, pyridyl, pyrazolyl, and thiazolyl.
 15. The compound of claim 1, wherein R₂ is selected from the group consisting of 2-furyl, 2-thienyl, 2-thiazolyl, 3-pyrazolyl, and 2-pyridyl.
 16. The compound of claim 1, wherein R₂ is 2-furyl.
 17. The compound of claim 1, wherein R₃ is hydrogen.
 18. The compound of claim 1, wherein R₄ is selected from H and substituted acyclic alkyl.
 19. The compound of claim 18, wherein R₄ is acyclic alkyl substituted by a compound selected from the group consisting of substituted aryl, unsubstituted aryl, cycloalkyl, non-aromatic heterocyclyl, CO₂R₅, CONR₆R₇, CONR₅NR₆R₇ and C(═NR₅)NR₆R₇.
 20. The compound of claim 19, wherein R₄ is acyclic alkyl substituted by substituted or unsubstituted aryl or CONR₆R₇.
 21. The compound of claim 20, wherein R₄ is methyl substituted by substituted or unsubstituted aryl or CONR₆R₇.
 22. The compound of claim 1, wherein R₄ is (CR₉R₁₀)_(n)R₁₁, wherein: n is 1 to 6, R₉ and R₁₀ are independently selected from H, alkyl and aryl, and R₁₁ is selected from the group consisting of substituted aryl, unsubstituted aryl, cycloalkyl, non-aromatic heterocyclic, CO₂R₅, CONR₆R₇, CONR₅NR₆R₇ and C(═NR₅)NR₆R₇.
 23. The compound of claim 22, wherein n is
 1. 24. The compound of claim 22, wherein R₉ and R₁₀ are independently selected from H and alkyl.
 25. The compound of claim 22, wherein R₁₁ is selected from substituted or unsubstituted aryl and CONR₆R₇.
 26. The compound of claim 22, wherein R₁₁ is a substituted aryl group represented by the formula Ar(R₁₁)_(a)(R₁₃)_(b)(R₁₄)_(c) wherein: Ar is an aryl group; R₁₂, R₁₃ and R₁₄ are substituent group(s), the same or different; and a, b and c are 0 or 1 such that a+b+c≧1.
 27. The compound of claim 26, wherein R₁₂, R₁₃ and R₁₄ are independently selected from the group consisting of NR₆R₇, alkyl, alkoxy, halogen, NO₂, CN, hydroxy, NHOH, CHO, CONR₆R₇, CO₂R₅, NR₅COR₆, NR₅CO₂R₈, NR₅SO₂R₈, OCO₂R₈ and aryl.
 28. The compound of claim 26, wherein R₁₂, R₁₃ and R₁₄ are independently selected from the group consisting of NR₆R₇, alkyl, and halogen.
 29. The compound of claim 27, wherein R₁₂, R₁₃, and R₁₄ are independently a substituted alkyl selected from the group consisting of alkoxyalkyl, hydroxyalkyl, aminoalkyl, and haloalkyl.
 30. The compound of claim 27, wherein R₁₂, R₁₃ and R₁₄ are independently selected from the group consisting of unsubstituted alkyl, NH₂, and fluoro.
 31. The compound of claim 19, wherein said substituted or unsubstituted aryl group is substituted or unsubstituted phenyl, thienyl, furyl, pyridyl or indolyl.
 32. The compound of claim 31, wherein said substituted or unsubstituted aryl group is substituted and unsubstituted phenyl, thienyl, furyl or pyridyl.
 33. The compound of claim 22, wherein R₁₁ is CONR₆R₇, R₆ is H, and R₇ is selected from the group consisting of H, unsubstituted alkyl, arylalkyl, and aryl.
 34. The compound of claim 1, wherein R₄ is CONR₆R₇, R₆ is H, and R₁ is arylalkyl.
 35. The compound of claim 34, wherein R₇ is arylmethyl.
 36. The compound of claim 1, wherein R₅ to R₈ are independently selected from lower alkyl.
 37. The compound of claim 1, wherein R₁ is NH₂, R₂ is 2-furyl, R₃ is H, and R₄ is arylmethyl.
 38. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of: (1) 6-chloro-4-(2-furyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine; (2) 4-(2-furyl)-N,N-dimethyl-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (3) 4-(2-furyl)-N-(2-hydroxyethyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (4) tert-butyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxylate; (5) 4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (6) 6-amino-N-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-carboxamide; (7) 1-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (8) 1-(3-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (9) 4-(2-furyl)-1-(4-nitrobenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (10) 1-(3-cyanobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (11) 1-(2-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (12) 4-(2-furyl)-1-(3-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (13) 4-(2-furyl)-1,N,N-tris(3-methoxybenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (14) 1-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (15) 4-(2-furyl)-1-(3-methylbenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (16) methyl 3-(6-amino-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidin-1-ylmethyl)benzoate; (17) 4-(2-furyl)-1-(4-sulphonylbenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (18) 4-(2-furyl)-1-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (19) 1-cyclohexylmethyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (20) 3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylmethyl)benzoic acid; (21) 4-(2-furyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (22) 4-(2-furyl)-1-(4-trifluoromethylbenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (23) 4-(2-furyl)-1-n-pentyl-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (24) 1-(2,6-difluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (25) 4-(2-furyl)-1-(3-pyridylmethyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (26) ethyl 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetate; (27) 6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetic acid; (28) 1-(2-fluorobenzyl)-4-(2-thienyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (29) 6-amino-4-(2-furyl)-N-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (30) 4-(2-furyl)-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (31) 4-(2-furyl)-1-(3-phenylpropyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (32) 1-(3-aminobenzyl)-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (33) 1-(2-fluorobenzyl)-4-(2-pyridyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (34) 6-amino-N-benzyl-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (35) 6-amino-4-(2-furyl)-N-phenyl-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (36) 6-amino-N-(3-chlorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (37) 6-amino-4-(2-furyl)-N-(2-furylmethyl)-1H-pyrazolo [3,4-d]pyrimidin-1-ylacetamide; (38) 1-(2-fluorobenzyl)-4-(2-thiazolyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (39) 4-(2-furyl)-1-(2-furylmethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (40) 4-(2-furyl)-1-(4-methylphenylsulphonyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (41) 1-(2-fluorobenzyl)-4-(1-methyl-2-imidazolyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (42) 1-(2-fluorobenzyl)-4-(2-imidazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (43) 6-amino-4-(2-furyl)-N-(3-methoxyphenyl)-1H-pyrazolo [3,4-d]pyrimidin-1-ylacetamide; (44) 6-amino-4-(2-furyl)-N-(2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (45) 6-amino-N-(2-fluorophenyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (46) 3-(6-amino-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidin-1-ylmethyl)-N-methylbenzamide; (47) 3-(6-amino-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidin-1-ylmethyl)-N,N-dimethylbenzamide; (48) 6-amino-4-(2-furyl)-N-(2-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (49) 6-amino-N-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidin-1-ylacetamide; (50) 1-(4-methylbenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (51) 1-(2-fluorobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (52) 4-(2-furyl)-1-(2-nitrobenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (53) N-(4-(2-furyl)-1-(3-(2-thiophenesulphonamido))benzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-yl)-2-thiophenesulphonamide; (54) N-((3-(6-amino-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-1-ylmethyl)phenyl-2-thiophenesulphonamide; (55) 1-(2-fluorobenzyl)-4-(3-pyrazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (56) 1-(2-aminobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (57) N-((3-(6-amino-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidine-1-ylmethyl)phenyl-1-methyl-1H-imidazol-4-sulphonamide; (58) 1-(3-nitrobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (59) 1-(3-nitrobenzyl)-4-(3-pyrazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (60) 1-(3-aminobenzyl)-4-(1,2,4-triazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (61) 1-(3-nitrobenzyl)-4-(2-thiazolyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (62) 1-(3-aminobenzyl)-4-(2-thiazolyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; (63) 1-(3-aminobenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (64) 1-(3-methoxybenzyl)-4-(1H-pyrazol-3-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; (65) 4-(2-furyl)-1-(4-indolylmethyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; and (66) tert-butyl 4-(6-amino-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidine-1-ylmethyl)indole-1-carboxylate.
 39. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of: 4-(2-furyl)-1-(3-methoxybenzyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; 1-(2-fluorobenzyl)-4-(2-furyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; 4-(2-furyl)-1-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; 1-(2,6-difluorobenzyl)-4-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine; 4-(2-furyl)-1-(3-phenylpropyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine; and 1-(2-fluorobenzyl)-4-(2-pyridyl)-1H-pyrazolo [3,4-d]pyrimidine-6-amine. 